||||||||III US005547842A United States Patent (19) 11 Patent Number: 5,547,842 Hogan Et Al

||||||||III US005547842A United States Patent (19) 11 Patent Number: 5,547,842 Hogan et al. (45) Date of Patent: Aug. 20, 1996

(54) NUCLEIC ACID PROBES FOR DETECTION FOREIGN PATENT DOCUMENTS SES..NTITATION OF NON-VIRAL 3138784 of 1985 Australia. O155359 9/1982 European Pat. Off.. (75) Inventors: James Hogan; Richard Smith, both of E. A: Eucal Eas g San Diego; Joann Kop, San Marcos, all 0.133671 7/1984 European Pat. Off.. of Calif. 0155360 9/1985 European Pat. Off. . 0232085 8/1987 European Pat. Off.. (73) Assignee: Gen-Probe Incorporated, San Diego, 0245129 11/1987 European Pat. Off.. Calif. 02772370250662 8/19881/1988 European Patoff.Pat. Off.. 8301073 3/1983 WIPO. (21) Appl. No.: 301,269 840,174 3/1984 WIPO. (22 Filed:tra. Sep. 6, 1994 88039578402721 7/19846/1988 WIPO . Related U.S. Application Data OTHER PUBLICATIONS 63 Continuation of Ser. No. 171,368, Dec. 21, 1993, aban- New England Biolab Catalog (1986/1987) (published by doned, which is a continuation of Ser. No. 907, 106, Jun. 26, New England Biolabs, Beverly MA, USA) p. 60. 1992, abandoned, which is a division of Ser. No. 806,929, Dcc. 11, 1991, abandoned, which is a continuation of Ser. No. 295,208, filed as PCT/US87/03009, Nov. 24, 1987, (List continued on next page.) abandoned, which is a continuation-in-part of Ser. No. 83,542, Aug. 7, 1987, abandoned, which is a continuation- Primary Examiner-W. Gary Jones in-part of Ser. No. 934,244, Nov. 24, 1986, abandoned. Attorney,Assistant Agent,Examiner or Firm-LyonArdin H. Marschel & Lyon (51) Int. Cl." ...... C12Q 1/68; C07H 21/04 52 U.S. Cl...... 435/6; 435/5; 435/91.1; (57) ABSTRACT 435/91.2, 435/810; 436/501; 536/22.1; A method for preparing probes, as well as several probes for 536/23.1536/24.1536/24.3: 536/24.31. - - 536/24.32; 536/24.33;s 935/77; 935/78 useico. in qualitative The i. or quantitative comprises hybridization OSG assaysal Gig are (58) Field of Search ...... 4355, 6,911 nucleotide that is sufficiently complementary to hybridize to 435/91.2, 810; 436/501; 536/22.1, 23.1, a region of rRNA selected to be unique to a non-viral 24.1, 24.3-24.33; 935/77, 78 organism or group of non-viral organisms sought to be detected, said region of rRNA being selected by comparing 56 References Cited one or more variable region rRNA sequences of said non viral organism or group of non-viral organisms with one or U.S. PATENT DOCUMENTS more variable region rRNA sequences from one or more 3,755,086 8/1973 Heimer ...... 195/103.5 R non-viral organisms sought to be distinguished. Hybridiza 3,930,956 l/1976 Juni ...... 195/103.5 R tion assay probes for Mycobacterium avium, Mycobacterium 4,033,143 7/1977 Juni ...... 195/100 intracellulare, the Mycobacterium tuberculosis-complex 4,228,238 10/1980 Swanson ...... 435/32 bacteria, Mycoplasma pneumoniae, Legionella, Salmonella, 4,237,224 12/1980 Cohen et al...... 435/68 Chlamydia trachomatis, Campylobacter, Proteus mirabilis, 4,275,149 6/1981 Litman et al...... 435/7 Enterococcus, Enterobacter cloacae, E. coli, Pseudomonas 4,302,204 11/1981 Wahl et al...... 23/230.2 group I, Neisseria gonorrhoeae, bacteria, and fungi also are

4,358,535 llll 982 Falkow et al...... 435/5 disclosed. 4,394,443 7/1983 Weissman et al...... 435/6 (List continued on next page.) 55 Claims, 28 Drawing Sheets

Summary of 23S rRNA Analysis -986 H------. 2-883 -HH------. -- 3-968 H------Ho-H------4-96.8 m--H------S-89 H------HHH------6-958 Hime-HHH ------7-92.7 ---4------...-H. 8-94. Herr Nitri------HHH-uh-whymn-H 99.4 +++++ - H ----- 0-90 H------1860 Hrith-----Hirt-firs------Hirii (2-858 ------HHH 3-739 E.coli-rif i to 20 so 46 so so is so so icon is so too is

LEGEND: SUMMARY OF 23S rRNA ANalysis (listiNG OF BACTEIA AND PERCENT SMARY INCLUDED NANAYSIS) 1986%. NeisseriA GolorRHoeAE-NESSERIA MENINGItois; 2.98.3% PROTEUS MRABS-proteus wuLGARIs, 3.96.8% MYCOBACTERIUM INTRACELLULARE-MYCOBACTERUMAWIUM; 4. 96.8% MycogACTERUMTBERCULCss-MycobacTERUMKANSASI;MYCOBACTERIUMAWU-MYCOBACTERUM ASEATCUM; 5, 95.9% 6.. 95.8% NKCOTIANA (ABACUM (TOBACCO)-2EA, MAS MAIZE); 7, 92.7% PROTEUs WULGARIS-KLessella RoscleroMATs; 8, 9.4% BACLUS STEAROTHERMCPHLS-Bacts SETILIS; 9, 9.4% MYCOBACTERIUMENTRACE.LULARE-MOEACTERIUMFORTUTUM, O, 9.0% ESCHERKCHIA COL-KLEBSELA RHINOSCLEROMATIS; I. 86.0% ESCHERICHA COL-PSEuccNAS AERUGINOSA; ta. 85.8% HAMYDIA TRACOMATIS-chLAMYCiA rst TAC; i3. 73.9% ESCHERKCHA COL-ANACYSTIs NoLANs, 5,547.842 Page 2

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Woese et al., “The Nucleotide Sequence of the 5S Riboso Yogev and Razin, "Common Deoxyribonucleic Acid mal RNA from a Photobacterium,' J. Mol. Evol. 5:35-46 Sequences in Mycoplasma genitalium and Mycoplasma (1975). Woese et al., "Secondary structure model for bacterial 16S pneumoniae Genomes,” Int'l J. System. Bacter: 35:426-430 ribosomal RNA: phylogenetic, enzymatic and chemical evi (1986). dence,' Nucleic Acids Research 8:2275–2293 (1980). Woese et al., "Sequence Characterization of 5S Ribosomal Yu et al., "Synthesis of Oligo- and Polynucleotides-XXI. RNA from Eight Gram Positive Procaryotes,” J. Mol. Evol. The Chemical Synthesis of Two Dodecanucleotides 8:143-153 (1976). Complementary to the 5'-Terminal Sequence of 16S rRNA Woese and Fox, "Methanogenic Bacteria.” Nature 273:101 (1978). of E. coli,” Bioorgan. Khimiya 5:1181-1900 (1979). Woese and Fox, "Phylogenetic Structure of the Prokaryotic Zablen, "Procaryotic Phylogeny by Ribosomal Ribonucleic Domain: The Primary Kingdoms,” Proc. Natl. Acad. Sci. USA 74:5088-5090 (1977). Acid Sequence Homology,” Microbiology, Dissertation Woese and Fox, "The Concept of Cellular Evolution,” J. Abstracts International, Nov. 1976, vol. 37, No. 5 p. 2083. Mol. Evol. 10:1-6 (1977). Wrede et al., "Binding Oligonucleotides to Escherichia coli Zablen et al., “Phylogenetic Origin of the Chloroplast and and Bacillus stearothermophilus 5S RNA." J. Mol. Biol. Prokaryotic Nature of Its Ribosomal RNA.' Proc. Natl. 120:83-96 (1978). Acad, Sci. USA 72:2418-2422 (1975). U.S. Patent Aug. 20, 1996 Sheet 1 of 28 5,547.842

A/G /.

U.S. Patent Aug. 20, 1996 Sheet 3 of 28 5,547.842

67/9/-/

OOI OO?2 OOº. OO!» OO9 OO9 OO?1 OO8 U.S. Patent Aug. 20, 1996 Sheet 4 of 28 5,547.842

A/G 2.

U.S. Patent Aug. 20, 1996 Sheet 5 of 28 5,547.842 U.S. Patent Aug. 20, 1996 Sheet 6 of 28 5,547.842

OOI OO2 OO2 OO!» OO9 OO9 OOZ OO8 OO6 OOOI

U.S. Patent Aug. 20, 1996 Sheet 9 of 28 5,547.842

A/G 3. U.S. Patent Aug. 20, 1996 Sheet 10 of 28 5,547.842

A/G 34.

E.coli UGCCUGGCGGCCGUAGCGCGGUGGUCCCACCUGACCCCAUGCCGAACUC Ecoli AGUAGGGAACUGCCAGGCAU U.S. Patent Aug. 20, 1996 Sheet 11 of 28 5,547.842

A/G 3A

AGAAGUGAAACGCCGUAGCGCCGAUGGUAGUGUGGGGUCUCCCCAUGCGAG 100 20 U.S. Patent Aug. 20, 1996 Sheet 12 of 28 5,547,842

A/G. 4.

A/G4E. U.S. Patent Aug. 20, 1996 Sheet 13 of 28 5,547,842

S. cerevisioe.sc UAUCUGGUUGAUCCUGCCAGUAGUCAUAUGCUUGUCUCAAA S.cerevisioe, sc UUAAAUCAGUUAUCGUUUAUUUGAUAGUUCCUUUACUACAU S.cerevisioe.sc GAUGUAUUUAUUAGAUAAAAAAUCAAUGUCUUCGCACUCUU S.cerevisioe.Sc AUUUCUGCCCUAUCAACUUUCGAUGGUAGGAUAGUGGCCUA S.cerevisionesc ACGGCUACCACAUCCAAGGAAGGCAGCAGGCGCGCAAAUUA S. Cerevisioe.Sc UUGUAAUUGGAAUGAGUACAAUGUAAAUACCUUAACGAGGA S.cerevisioe.sc AUUAAAGUUGUUGCAGUUAAAAAGCUCGUAGUUGAACUUUG S.cerevisioe.Sc UGGCUAACCUUGAGUCCUUGUGGCUCUUGGCGAACCAGGAC S. Cerevisioe.Sc GGAAUAAUAGAAUAGGACGUUUGGUUCUAUUUUGUUGGUUU S.cerevisioe.Sc GUGAAAUUCUUGGAUUUAUUGAAGACUAACUACUGCGAAAG S.cerevisioe, sc GAUACCGUCGUAGUCUUAACCAUAAACUAUGCCGACUAGGG S.cerevisioe.Sc GUUCUGGGGGGAGUAUGGUCGCAAAGGCUGAAACUUAAAGG S.cerevisioe.sc GAAACUCACCAGGUCCAGACACAAUAAGGAUUGACAGAUUG S.cerevisioe.SC AUUUGUCUGCUUAAUUGCGAUAACGAACGAGACCUUAACCU S.cerevisioe.Sc AAGCCGAUGGAAGUUUGAGGCAAUAACAGGUCUGUGAUGCC S.cerevisioe.Sc GCCGAGAGGUCUUGGUAAUCUUGUGAAACUCCGUCGUGCUG S.Cerevisioe.Sc CAGCUUGCGUUGAUUACGUCCCUGCCCUUUGUACACACCGC S.cerevisioe.sc GCAACUCCAUCUCAGAGCGGAGAAUUUGGACAAACUUGGUC S.cerevisioe.sc UA

A/G 44. U.S. Patent Aug. 20, 1996 Sheet 14 of 28 5,547.842

GAUUAAGCCAUGCAUGUCUAAGUAUAAGCAAUUUAUACAGUGAAACUGCGAA GGUAUAACCGUGGUAAUUCUAGAGCUAAUACAUGCUUAAAAUCUCGACCCUU UGAUGAUUCAUAAUAACUUUUCGAAUCGCAUGGCCUUGUGCUGGCGAUGGUU CCAUGGUUUCAACGGGUAACGGGGAAUAAGGGUUCGAUUCCGGAGAGGGAGC CCCAAUCCUAAUUCAGGGAGGUAGUGACAAUAAAUAACGAUACAGGGCCCAU ACAAUUGGAGGGCAAGUCUGGUGCCAGCAGCCGCGGUAAUUCCAGCUCCAAU GGCCCGGUUGGCCGGUCCGAUUUUUUCGUGUACUGGAUUUCCAACGGGGCCU UUUUACUUUGAAAAAAUUAGAGUGUUCAAAGCAGGCGUAUUGCUCGAAUAUA CUAGGACCAUCGUAAUGAUUAAUAGGGACGGUCGGGGGCAUCGGUAUUCAAU CGUUUGCCAAGGACGUUUUCGUUAAUCAAGAACGAAAGUUGAGGGAUCGAAG AUCGGGUGGUGUUUUUUUAAUGACCCACUCGGUACCUUACGAGAAAUCAAAG AAUUGACGGAAGGGCACCACCAGGAGUGGAGCCUGCGGCUUAAUUUGACUCA AGAGCUCUUUCUUGAUUUUGUGGGUGGUGGUGCAUGGCCGUUUCUCAGUUGG ACUAAAUAGUGGUGCUAGCAUUUGCUGGUUAUCCACUUCUUAGAGGGACUAU CUUAGAACGUUCUGGGCCGCACGCGCGCUACACUGACGGAGCCAGCGAGUCU GGGAUAGAGCAUUGUAAUUAUUGCUCUUCAACGAGGAAUUCCUAGUAAGCGC CCGUCGCUAGUACCGAUUGAAUGGCUUAGUGAGGCCUCAGGAUCUGCUUAGA AUUUGGAGGAACUAAAAGUCGUAACAAGGUUUCCGUAGGUGAACCUGCGGAA

A/G 4A U.S. Patent Aug. 20, 1996 Sheet 15 of 28 5,547.842

UGGCUCA OO UGGAAGA 20O CAUUCAA. 3OO CUGAGAA 4OO UCGGGUC 5OO AGCGUAU 6OO UUCCUUC 7OO UUAGCAU 8OO UGUCGAG 90O ACGAUCU IOOO A/G. 4C UCUUUGG OO ACACGGG i2OO UGGAGUG 3OO CGGUUUC 4OO AACCUUG 5OO AAGUCAU 6OO GAAGGGG 7OO GGAUCAU 8OO 1802 U.S. Patent Aug. 20, 1996 Sheet 16 of 28 5,547.842

A/G 5.

U.S. Patent Aug. 20, 1996 Sheet 17 of 28 5,547.842 FIG. 5A

S.cerevisioe AAACUUUCAACAACGGAUCUCUUGGUUCUCGCAUCGAUGAAGA S.cerevisioe UUGGUAUUCCAGGGGGCAUGCCUGUUUGAGCGUCAUUUGUUUG Scerevisioe UAGUAACGGCGAGUGAAGCGGCAAAAGCUCAAAUUUGAAAUCU Scerevisioe ACGUCAUAGAGGGUGAGCAUCCCGUGUGGCGAGGAGUGCGGUU Scerevisioe AAUAUUGGCGAGAGACCGAUAGCGAACAAGUACAGUGAUGGAA Scerevisioe UGUUUUGUGCCCUCUGCUCCUUGUGGGUAGGGGAAUCUCGCAU S.cerevisioe GUGGGAAUACUGCCAGCUGGGACUGAGGACUGCGACGUAAGUC Scerevisioe GGGUGUAAAACCCAUACGCGUAAUGAAAGUGAAC GUAGGUUGG Scerevisione cGAAAGAUGGUGAACUAUGCCU GAAUAGGGUGAAGCCAGAGGA Scerevisioe GAACCAUCUAGUAGCUGGUUCCUGCCGAAGUUUCCCUCAGGAU Scerevisione UCAAACUUUAAAUAUGUAAGAAGUCCUUGUUACUUAAUUGAAC Scerevisioe GAGUUAAGGUGCCGGAAUACACGCUCAUCAGACACCACAAAAG Scerevisione CGAAUGAACUAGCCCUGAAAAUGGAUGGCGCUCAAGCGUGUUA Scerevisioe GUAAGGUCGGGUCGAACGGCCUCUAGUGCAGAUCUUGGUGGUA Scerevisioe AGUCGAUCCUAAGAGAUGGGGAAGCUCCGUUUCAAAGGCCUGA S.cerevisione UGAAUGUGGAGACGUCGGCGCGAGCCCUGGGAGGAGUUAUCUU S.cerevisioe UGCUGGCUCCGGUGCGCUUGUGACGGCCCGUGAAAAUCCACAG Scerevisiae UAAUGUAGAUAAGGGAAGUCGGCAAAAUAGAUCCGUAACUUCG Scerevisione GGGGCUUGCUCUGCUAGGCGGACUACUUGCGUGCCUUGUUGUA S.cerevisioe GAAUCUGACUGUCUAAUUAAAACAUAGCAUUGCGAUGGUCAGA Scerevisione ACGGCGGGAGUAACUAUGACUCUCUUAAGGUAGCCAAAUGCCU Scerevisioe GCCAAGGGAACGGGCUUGGCAGAAUCAGCGGGGAAAGAAGACC U.S. Patent Aug. 20, 1996 Sheet 18 of 28 5,547.842

FIG 5B.

ACGCAGCGAAAUGCGAUACGUAAUGUGAAUUGCAGAAUUCCGUGAAUCAUCG ACCUCAAAUCAGGUAGGAGUACCCGCUGAACUUAAGCAUAUCAAUAAGCGGA GGUACCUUCGGUGCCCGAGUUGUAAUUUGGAGAGGGCAACUUUGGGGCCGUU CUUUGUAAAGUGCCUUCGAAGAGUCGAGUUGUUUGGGAAUGCAGCUCUAAGU AGAUGAAAAGAACUUUGAAAAGAGAGUGAAAAAGUACGUGAAAUUGUUGAAA UUCACUGGGCCAGCAUCAGUUUUGGUGGCAGGAUAAAUCCAUAGGAAUGUAG AAGGAUGCUGGCAUAAUGGUUAUAUGCCGCCCGUCUUGAAACACGGACCAAG GGCCUCGCAAGAGGUGCACAAUCGACCGAUCCUGAUGUCUUCGGAUGGAUUU AACUCUGGUGGAGGCUCGUAGCGGUUCUGACGUGCAAAUCGAUCGUCGAAUU AGCAGAAGCUCGUAUCAGUUUUAUGAGGUAAAGCGAAUGAUUAGAGGUUCCG GUGGACAUUUGAAUGAAGAGCUUUUAGUGGGCCAUUUUUGGUAAGCAGAACU GUGUUAGUUCAUCUAGACAGCCGGACGGUGGCCAUGGAAGUCGGAAUCCGCU CCUAUACUCUACCGUCAGGGUUGAUAUGAUGCCCUGACGAGUAGGCAGGCGU GUAGCAAAUAUUCAAAUGAGAACUUUGAAGACUGAAGUGGGGAAAGGUUCCA UUUUAUGCAGGCCACCAUCGAAAGGGAAUCCGGUAAGAUUCCGGAACUUGGA UUCUUCUUAACAGCUUAUCACCCCGGAAUUGGUUUAUCCGGAGAUGGGGUCU GAAGGAAUAGUUUUCAUGCUAGGUCGUACUGAUAACCGCAGCAGGUCUCCAA GGAUAAGGAUUGGCUCUAAGGGUCGGGUAGUGAGGGCCUUGGUCAGACGCAG GACGGCCUUGGUAGGUCUCUUGUAGACCGUCGCUUGCUACAAUUAACAGAUC AAGUGAUGUUGACGCAAUGUGAUUUCUGCCCAGUGCUCUGAAUGUCAAAGUG CGUCAUCUAAUUAGUGACGCGCAUGAAUGGAUUAACGAGAUUCCCACUGUCC CUGUUGAGCUUGACUCUAGUUUGACAUUGUGAAGAGACAUAGAGGGUGUAGA U.S. Patent Aug. 20, 1996 Sheet 19 of 28 5,547.842

AAUCUUUGAACGCACAUUGCGCCCC i2O GGAAAAGAAACCAACCGGAUUGCCU 240 CCUUGUCUAUGUUCCUUGGAACAGG 36O GGGUGGUAAAUUCCAUCUAAAGCUA 48O GGGAAGGGCAUUUGAUCAGACAUGG 6OO CUUGCCUCGGUAAGUAUUAUAGCCU 720 GAGUCUAACGUCUAUGCGAGUGUUU 840 GAGUAAGAGCAUAGCUGUUGGGACC 96.O UGGGUAUAGGGGCGAAAGACUAAUC O8O GGGUCGAAAUGACCUUGACCUAUUC I2OO F.IG. 5C. GGCGAUGCGGGAUGAACCGAACGUA 320 AAGGAGUGUGUAACAACUCACCGGC 440 GGAGGUCAGUGACGAAGCCUAG ACC 56O CGUCAACAGCAGUUGGACGUGGGUU 68O UAUGGAUUCUUCACGGUAACGUAAC 8OO UAUGGCUGGAAGAGGCCAGCACCUU 920 GGUGAACAGCCUCUAGUUGAUAGAA 204O CGGGCGUGCUUGUGGACUGCUUGGU 26O AACUUAGAACUGGUACGGACAAGGG 228O AAGAAAUUCAACCAAGCGCGAGUAA 24OO CUAUCUACUAUCUAGCGAAACCACA 252O AUAAGUGGGAGCUUCGGCGCCAGUG 264O U.S. Patent Aug. 20, 1996 Sheet 20 of 28 5,547.842

S.cerevisioe AAAUACCACUACCUUUAUAGUUUCUUUACUUAUUCAAUGAAGC Scerevisioe UGGGGAGUUUGGCUGGGGCGGCACAUCUGUUAAACGAUAACGC Scerevisione GUGUGAAUACAAACCAUUGAAAGUGUGGCCUAUCGAUCCUUUA S.cerevisione AGCGACAUUGCUUUUUGAUUCUUCGAUGUCGGCUCUUCCUAUC Scerevisione AGACAGGUUAGUUUUACCCUACUGAUGAAUGUUACCAGCAAUA Scerevisioe AAGCACCAUCCGCUGGAUUAUGGCUGAACGCCUCUAAGUCAGA S.cerevisione UGAACCAUAGCAGGCUAGCAACGGUGCACUUGGCGGAAAGGCC S.cerevisione GGUAUUGUAAGCGGUAGAGUAGCCUUGUUGUUACGAUCUGCUG Scerevisiae S.cerevisione

FIG 5D. U.S. Patent Aug. 20, 1996 Sheet 21 of 28 5,547.842

GGAGCUGGAAUUCAUUUUCCACGUUCUAGCAUUCAAGGUCCCAUUCGGGGCU AGAUGUCCUAAGGGGGGCUCAUGGAGAACAGAAAUCUCCAGUAGAACAAAAG GUCCCUCGGAAUUUGAGGCUAGAGGUGCCAGAAAAGUUACCACAGGGAUAAC AUACCGAAGCAGAAUUCGGUAAGCGUUGGAUUGUUCACCCACUAAUAGGGAA GUAAUUGAACUUAGUACGAGAGGAACAGUUCAUUCGGAUAAUUGGUUUUUGC AUCCAUGCUAGAACGCGGUGAUUUCUUUGCUCCACACAAUAUAGAUGGAUAC UUGGGUGCUUGCUGGCGAAUUGCAAUGUCAUUUUGCGUGGGGAUAAAUCAUU AGAUUAAGCCUUUGUUGUCUGAUUUGU

FIG 5E. U.S. Patent Aug. 20, 1996 Sheet 22 of 28 5,547.842

GAUCCGGGUUGAAGACAUUGUCAGG 276O GGUAAAGCCCCUUAGUUUGAUUUCA 288O UGGCUUGUGGCAGUCAAGCGUUCAU CAUGAGCUGGGUUUAGACCGUCGUG 32O GGCUGUCUGAUCAGGCAUUGCCGCG 324O GAAUAAGGCGUCCUUGUGGCGUCGC 336O UGUAUACGACUUAGAUGUACAACGG 348O 355O

355O 355o

FIG 5F U.S. Patent Aug. 20, 1996 Sheet 23 of 28 5,547.842

Summary of 16S rRNA Analysis

E. coli-ref l00 200 300 400 500 600 700 800 900 000 100 1200 300 400 500

LEGEND: SUMMARY OF 16S rRNA ANALYSIS (LISTING OF BACTERIA AND PERCENT SIMLARITY INCLUDED IN ANALYSS) l. 99.7% CLOSTRIDIUM BOTULINUM G-CLOSTRIDIUM SUBTERMINALE; 2. 99.4% STREPTOCOCCUS CREMORIS-STREPTOCOCCUS LACTIS; 3. 99.1% LACTOBACILLUS LACTIS LACTOBACILLUS DELBRUECKl; 4.99.0% NEISSERLA GONORRHOEAE-NEISSERIA MENINGITIDIS; 5.99.0% MYCOBACTERIUN INTRACELLULARE MYCOBACTERIUM AVIUM; 6.. 98.4% MYCOBACTERIUM AVIUM-MYCOBACTERIUM TUBERCULOSIS; 7. 97.3% PSEUDOMONAS ALCALIGENES-PSEUDOMONAS STUTZERI; 8. 95.6% CHLAMYDIA PSITTAC-CHLAMYDIA TRACHOMATIS; 9. 95.5% SPIROPLASMA CITRi-SPIROPLASMA MIRUM; IO. 94.0% CLOSTRIDIUM LITUSEBURENSE-CLOSTRIDIUM SODELLI; i. 93.3% LISTERIA MONOCYTOGENES-BROCHOTHRIX THERMOSPHACTA, 12. 73.7% ESCHERICHA COL BACTERODES FRAGILS.

FIG 6. U.S. Patent Aug. 20, 1996 Sheet 24 of 28 5,547.842

Summary of 23S rRNA Analysis

-986 2-98.3 3-96.8 4-96.8 5-95.9 6-95.8 7-92.7 8-91.4 9-9.4 0-9.0 -86.0 2-858 3-739 E.coli-ref 00 200 300 400 500 600 700 800 900 000 00 200 300 400 1500

LEGEND: SUMMARY OF 23S rRNA ANALYSIS (LISTING OF BACTEIA AND PERCENT SMILARITY INCLUDED IN ANALYSIS) l, 98.6% NEISSERIA GONORRHOEAE-NEISSERIA MENINGITIDIS; 2. 98.3% PROTEUS MIRABILIS-PROTEUS VULGARIS; 3. 96.8% MYCOBACTERIUM INTRACELLULARE-MYCOBACTERIUM AVIUM; 4. 96.8% MYCOBACTERIUM AVIUM-MYCOBACTERIUM ASIATICUM; 5. 95.9% MYCOBACTERIUM TUBERCULOSIS-MYCOBACTERIUM KANSASI; 6.. 95.8% NICOTIANA TABACUM (TOBACCO)-ZEA MAYS (MAIZE); 7. 92.7% PROTEUS VULGARIS-KLEBSIELLA RHINOSCLEROMATIS; 8, 9.4% BACILLUS STEAROTHERMOPHILUS-BACILLUS SUBTILIS; 9. 9.4% MYCOBACTERIUM INTRACELLULARE-MYCOBACTERIUM FORTUITUM, IO. 91.0% ESCHERICHA COL-KLEBSIELLA RHINOSCLEROMATIS ; II. 86.0% ESCHERICHIA COL-PSEUDOMONAS AERUGINOSA; 12. 85.8% CHLAMYDIA TRACHOMATIS-CHLAMYDIA PSITTACl; 13. 73.9% ESCHERICHA COL-ANACYSTS NDULANS.

FIG 7 U.S. Patent Aug. 20, 1996 Sheet 25 of 28 5,547.842

Summary of 23S rRNA Analysis

E.coli-ref ISO 600 700 800 900 2000 200 2200 2300 2400 2500 2600 2700 2800 2900 LEGEND: SUMMARY OF 23S rRNA ANALYSIS (LISTING OF BACTEA AND PERCENT SIMLARITY INCLUDED IN ANALYSIS) , 98.6% NESSERIA GONORRHOEAE-NEISSERIA MENINGTDIS; 2. 98.3% PROTEUS MIRABLIS-PROTEUS VULGARIS; 3. 96.8% MYCOBACTERIUM INTRACELLULARE-MYCOBACTERIUM AVIUM; 4. 96.8% MYCOBACTERIUM AVIUM-MYCOBACTERIUM ASIATICUM; 5. 95.9% MYCOBACTERIUM TUBERCULOSIS-MYCOBACTERIUM KANSASl; 6.. 95.8% NCOTIANA TABACUM (TOBACCO)-ZEA MAYS (MAIZE); 7. 92.7% PROTEUS VULGARIS-KLEBSIELLA RHINOSCLEROMATS; 8. 91.4% BACILLUS STEAROTHERMOPHILUS-BACILLUS SUBTILIS; 9. 91.4% MYCOBACTERIUM INTRACELLULARE-MYCOBACTERIUM FORTUITUM, O. 9.O% ESCHERICHA COL-KLEBSIELLA RHINOSCLEROMATIS ; 86.0% ESCHERICHA COL-PSEUDOMONAS AERUGINOSA; 2. 85.8% CHLAMYDIA TRACHOMATIS-CHLAMYDIA PSITTACl; 3. 73.9% ESCHERICHA COL-ANACYSTS NDULANS.

FIG 8. U.S. Patent Aug. 20, 1996 Sheet 26 of 28 5,547.842

VNHIS9||?oÁSuo?e00||HGOHd 00900200800100||,000|006 JeuJuunS

5,547,842 1 2 NUCLECACD PROBES FOR DETECTION Viruses,' filed Sep. 4, 1984 (Kohne II), both of which are AND/OR QUANTITATION OF NON-VIRAL incorporated by reference, together with all other applica ORGANISMS tions cited herein. Also described in those applications are methods for This is a continuation of Hogan et al., U.S. Ser. No. 5 determining the presence of RNA-containing organisms in a 08/171,368, filed Dec. 21, 1993, now abandoned, which is a sample which might contain such organisms, comprising the continuation of Hogan et al., U.S. Ser. No. 07/907,106, filed steps of bringing together any nucleic acids from a sample Jun. 26, 1992, now abandoned, which is a divisional of and a probe comprising nucleic acid molecules which are Hogan et al., U.S. Ser. No. 07/806,929, filed Dec. 11, 1991, shorter than the rRNA subunit sequence from which it was now abandoned, which is a continuation of Hogan et al., 10 derived and which are sufficiently complementary to hybrid U.S. Ser. No. 07/295,208, filed Dec. 9, 1988, now aban ize to the rRNA of one or more non-vital organisms or doned, which is the national filing of Hogan et al., PCT/ groups of non-vital organisms, incubating the mixture under US87/03009, filed Nov. 24, 1987, which is a continuation specified hybridization conditions, and assaying the result in-part of Hogan et al., U.S. Ser. No. 07/083,542, filed Aug. ing mixture for hybridization of the probe and any test 7, 1987, now abandoned, which is a continuation-in-part of 15 sample rRNA. The invention is described to include using a Hogan et al., U.S. Ser. No. 06/934,244, filed Nov. 24, 1986, probe which detects only rRNA subunit subsequences which now abandoned. are the same or sufficiently similar in particular organisms or groups of organisms and is said to detect the presence or BACKGROUND OF THE INVENTION absence of any one or more of those particular organisms in 20 a sample, even in the presence of many non-related organ 1. Field of the Invention SS. The inventions described and claimed herein relate to We have discovered and describe herein a novel method probes and assays based on the use of genetic material such and means for designing and constructing DNA probes for as RNA. More particularly, the inventions relate to the use in detecting unique rRNA sequences in an assay for the design and construction of nucleic acid probes and hybrid 25 detection and/or quantitation of any group of non-vital ization of such probes to genetic material of target non-vital organisms. Some of the inventive probes herein may be used organisms in assays for detection and/or quantitation thereof to detect and/or quantify a single species or strain of in test samples of, e.g., sputum, urine, blood and tissue non-viral organism and others may be used to detect and/or sections, food, soil and water. quantify members of an entire genus or desired phylogenetic 2. Introduction 30 grouping. Two single strands of nucleic acid, comprised of nucle otides, may associate ("hybridize") to form a double helical structure in which the two polynucleotide chains running in SUMMARY OF THE ENVENTION opposite directions are held together by hydrogen bonds (a 35 In a method of probe preparation and use, a single strand weak form of chemical bond) between pairs of matched, deoxyoligonucleotide of particular sequence and defined centrally located compounds known as "bases.” Generally, length is used in a hybridization assay to determine the in the double helical structure of nucleic acids, for example, presence or amount of rRNA from particular target non-viral the base adenine (A) is hydrogen bonded to the base thymine organisms to distinguish them from their known closest (T) or uracil (U) while the base guanine (G) is hydrogen 40 phylogenetic neighbors. Probe sequences which are specific, bonded to the base cytosine (C). At any point along the respectively, for 16S rRNA variable subsequences of Myco chain, therefore, one may find the base pairs AT or AU, TA bacterium avium, Mycobacterium intracellulare and the or UA, GC, or CG. One may also find AG and GU base pairs Mycobacterium tuberculosis-complex bacteria, and which in addition to the traditional ("canonical') base pairs. do not cross react with nucleic acids from each other, or any Assuming that a first single strand of nucleic acid is suffi 45 other bacterial species or respiratory infectious agent, under ciently complementary to a second and that the two are proper stringency, are described and claimed. A probe spe brought together under conditions which will promote their cific to three 23S rRNA variable region subsequences from hybridization, double stranded nucleic acid will result. the Mycobacterium tuberculosis-complex bacteria is also Under appropriate conditions, DNA/DNA, RNA/DNA, or described and claimed, as are rRNA variable region probes RNA/RNA hybrids may be formed. 50 useful in hybridization assays for the genus Mycobacterium Broadly, there are two basic nucleic acid hybridization (16S 23S rRNA specific), Mycoplasma pneumoniae (5S and procedures. In one, known as "in solution” hybridization, 16S rRNA-specific), Chlamydia trachomatis (16S and 23S both a "probe' nucleic acid sequence and nucleic acid rRNA specific), Enterobacter cloacae (23S rRNA specific), molecules from a test sample are free in solution. In the other Escherichia coli (16S rRNA specific), Legionella (16S and method, the sample nucleic acid is usually immobilized on 55 23S rRNA specific), Salmonella (16S and 23S rRNA spe a solid support and the probe sequence is free in solution. cific), Enterococci (16S rRNA specific), Neisseria gonor A probe may be a single strand nucleic acid sequence rhoeae (16S rRNA specific), Campylobacter (16S rRNA which is complementary in some particular degree to the specific), Proteus mirabilis (23S rRNA specific), Pseudomo nucleic acid sequences sought to be detected ("target nas (23S rRNA specific), fungi (18S and 28S rRNA spe sequences'). It may also be labelled. A background descrip 60 cific), and bacteria (16S and 23S rRNA specific). tion of the use of nucleic acid hybridization as a procedures In one embodiment of the assay method, a test sample is for the detection of particular nucleic acid sequences is first subjected to conditions which release rRNA from any described in U.S. application Ser. No. 456,729, entitled non-viral organisms present in that sample. rRNA is single "Method for Detection, Identification and Quantitation of stranded and therefore available for hybridization with suf Non-Viral Organisms,” filed Jan. 10, 1983 (Kohne I), and 65 ficiently complementary genetic material once so released. U.S. application Ser. No. 655,365, entitled "Method For Contact between a probe, which can be labelled, and the Detecting, Identifying and Quantitating Organisms and rRNA target may be carried out in solution under conditions 5,547,842 3 4 which promote hybridization between the two strands. The probe may be an oligonucleotide or a nucleotide poly reaction mixture is then assayed for the presence of hybrid her. ized probe. Numerous advantages of the present method for hybrid: the complex formed between two single stranded the detection of non-vital organisms over prior art tech nucleic acid sequences by Watson-Crick base pairings niques, including accuracy, simplicity, economy and speed or non-canonical base pairings between the comple will appear more fully from the detailed description which mentary bases. follows. hybridization: the process by which two complementary strands of nucleic acids combine to form double BRIEF DESCRIPTION OF THE DRAWING stranded molecules (hybrids). FIG. 1 is a chart of the primary structure of bacterial 16S 10 complementarity: a property conferred by the base rRNA for Escherichia coli, depicting standard reference sequence of a single strand of DNA or RNA which may numbers for bases. form a hybrid or double stranded DNA:DNA, RNA:RNA or DNA:RNA through hydrogen bonding FIG. 2 is a chart of the primary structure of bacterial 23S between Watson-Crick base pairs on the respective rRNA for Escherichia coli, depicting standard reference 15 strands. Adenine (A) usually complements thymine (T) numbers for bases. or Uracil (U), while guanine (G) usually complements FIG. 3 is a chart of the primary structure of bacterial 5S cytosine (C). rRNA for Escherichia coli, depicting standard reference stringency: term used to describe the temperature and numbers for bases. solvent composition existing during hybridization and FIG. 4 is a chart of the primary structure for the 18S rRNA 20 the subsequent processing steps. Under high stringency for Saccharomyces cerevisiae, depicting standard reference conditions only highly homologous nucleic acid numbers for bases. hybrids will form; hybrids without a sufficient degree FIG. 5 is a chart of the primary structure for the 28S rRNA of complementarity will not form. Accordingly, the for Saccharomyces cerevisiae, depicting standard reference stringency of the assay conditions determine the numbers for bases. 25 amount of complementarity needed between two FIG. 6 is a diagram showing the locations in the 16S nucleic acid strands forming a hybrid. Stringency is rRNA (using E. coli reference numbers) which differ chosen to maximize the difference in stability between bertween 12 different sets of related organisms. In Example the hybrid formed with the target and the nontarget 1, for example, 99.7 refers to the difference in 16S rRNA nucleic acid. between Clostridium botuliniumg and Clostridium subter 30 probe specificity: characteristic of a probe which minale. describes its ability to distinguish between target and FIG. 7 is a diagram showing the locations in the first 1500 nontarget sequences. Dependent on sequence and assay bases of 23S rRNA (using E.coli reference numbers) which conditions. Probe specificity may be absolute (i.e., differ between 12 different sets of related organisms. 35 probe able to distinguish between target organisms and FIG. 8 is a diagram showing the locations in the terminal any nontarget organisms), or it may be functional (i.e., bases of 23S rRNA (using E.coli reference numbers) which probe able to distinguish between the target organism differ between 12 different sets of related organisms. and any other organism normally present in a particular FIG. 9 is a schematic representation of the location of sample). Many probe sequences can be used for either 40 broad or narrow specificity depending on the conditions probes capable of hybridizing to the 16S rRNA. of use. FIG. 10 is a schematic representation of the location of variable region: nucleotide polymer which differs by at probes capable of hybridizing to the first 1500 bases of the least one base between the target organism and non 23S rRNA. target organisms contained in a Sample. FIG. 11 is a schematic representation of the location of probes capable of hybridizing to the terminal bases of 23S 45 conserved region: a region which is not variable. rRNA. sequence divergence: process by which nucleotide poly mers become less similar during evolution. DETAILED DESCRIPTION OF THE INVENTION sequence convergence: process by which nucleotide poly mers become more similar during evolution. Definitions 50 The following terms, as used in this disclosure and claims, bacteria: members of the phylogenetic group eubacteria, are defined as: which is considered one of the three primary kingdoms. nucleotide: a subunit of a nucleic acid consisting of a Tm: temperature at which 50% of the probe is converted phosphate group, a 5' carbon sugar and a nitrogen from the hybridized to the unhybridized form. containing base. In RNA the 5' carbon sugar is ribose. 55 thermal stability: Temperature at which 50% of the pro In DNA, it is a 2-deoxyribose. The term also includes be:target hybrids are converted to the single stranded analogs of such subunits. form. Factors which affect the thermal stability can affect probe specificity and therefore, must be con nucleotide polymer: at least two nucleotides linked by trolled. Whether a probe sequence is useful to detect phosphodiester bonds. 60 only a specific type of organism depends largely on the oligonucleotide: a nucleotide polymer generally about 10 thermal stability difference between probe:target to about 100 nucleotides in length, but which may be hybrids (“P:T") and probe:nontarget hybrids (“P:NT"). greater than 100 nucleotides in length. In designing probes the Tm P:T minus the Tm P:NT nucleic acid probe: a single stranded nucleic acid should be as large as possible. sequence that will combine with a complementary 65 In addition to a novel method for selecting probe single stranded target nucleic acid sequence to form a sequences, we have discovered that it is possible to create a double-stranded molecule (hybrid). A nucleic acid DNA probe complementary to a particular rRNA sequence 5,547,842 5 6 obtained from a single type of target microorganism and to amounts of similarity between them. Closer analysis of these successfully use that probe in a non-cross reacting assay for figures reveals some subtle patterns between these closely the detection of that single microorganism, even in the related organisms. In all cases studied, we have seen suffi presence of its known, most closely related taxonomic or cient variation between the target organism and the closest phylogenetic neighbors. With the exception of viruses, all phylogenetic relative found in the same sample to design the prokaryotic organisms contain rRNA molecules including probe of interest. Moreover, in all cases studied to date, the 5S rRNA, 16S rRNA, and a larger rRNA molecule known as per cent similarity between the target organism (or organ 23S rRNA. Eukaryotes are known to have 5.0S, 5.8S, 18S isms) and the closest phylogenetically related organisms and 28S rRNA molecules or analogous structures. (The term found in the same sample has been between 90% and 99%. "16S like' sometimes is used to refer to the rRNA found in Interestingly, there was enough variation even between the the small ribosomal subunit, including 18S and 17S rRNA. 10 rRNA's of Neisseria's gonorrhoeae and meningitidis (See Likewise the term '23S like' rRNA sometimes is used to Example 21) to design probes- despite the fact that refer to the rRNA found in the large ribosomal subunit. 5.8S DNA:DNA homology studies suggested these two species rRNA is equivalent to the 5' end of the 23S like rRNA) might actually be one and the same. These rRNA molecules contain nucleotide sequences which These figures also show that the differences are distrib are highly conserved among all organisms thus far exam 15 uted across the entire 16S and 23S rRNA's. Many of the ined. There are known methods which allow a significant differences, nonetheless, cluster into a few regions. These portion of these rRNA sequences to be determined. For locations in the rRNA are good candidates for probe design, example, complementary oligonucleotide primers of about with our current assay conditions. We also note that the 20–30 bases in length can be hybridized to universally locations of these increased variation densities usually are conserved regions in purified rRNA that are specific to the 20 situated in the same regions of the 16S and 23S rRNA for 5S, 16S, or 23S subunits and extended with the enzyme comparable per cent similarity values. In this manner, we reverse transcriptase. Chemical degradation or dideoxy have observed that certain regions of the 16S and 23S rRNA nucleotide- terminated sequencing reactions can be used to are the most likely sites in which significant variation exists determine the nucleotide sequence of the extended product. between the target organism and the closest phylogenetic Lane, D.J. et al., Proc. Nat'l Acad. Sci. USA 82,6955–6959 25 relatives found in a sample. We have disclosed and claimed (1985). species specific probes which hybridize in these regions of In our invention, comparison of one or more sequenced significant variation between the target organism and the rRNA variable regions from a target organism to one or closest phylogenetic relative found in a sample. more rRNA variable region sequences from a closely related FIGS. 9, 10 and 11 are a schematic representation of the bacterial species is utilized to select a sequence unique to the 30 location of probes disclosed and claimed herein. Because rRNA of the target organism. rRNA is preferable to DNA as 16S and 23S RNAs do not, as a rule, contain sequences of a probe target because of its relative abundance and stability duplication longer than about six nucleotides in length, in the cell and because of its patterns of phylogenetic probes designed by these methods are specific to one or a conservation. few positions on the target nucleic acid. Notwithstanding the highly conserved nature of rRNA, 35 The sequence evolution at each of the variable regions we have discovered that a number of regions of the rRNA (for example, spanning a minimum of 10 nucleotides) is, for molecule which can vary in sequence, can vary even the most part divergent, not convergent. Thus, we can between closely related species and can, therefore, be uti confidently design probes based on a few rRNA sequences lized to distinguish between such organisms. Differences in which differ between the target organism and its phyloge the rRNA molecule are not distributed randomly across the 40 netically closest relatives. Biological and structural con entire molecule, but rather are clustered into specific straints on the rRNA molecule which maintain homologous regions. The degree of conservation also varies, creating a primary, secondary and tertiary structure throughout evolu unique pattern of conservation across the ribosomal RNA tion, and the application of such constraints to probe diag subunits. The degree of variation and the distribution nostics is the subject of ongoing study. The greater the thereof, can be analyzed to locate target sites for diagnostic 45 evolutionary distance between organisms, the greater the probes. This method of probe selection may be used to select number of variable regions which may be used to distinguish more than one sequence which is unique to the rRNA of a the organisms. target organism. Once the variable regions are identified, the sequences are We have identified variable regions by comparative analy aligned to reveal areas of maximum homology or "match'. sis of rRNA sequences both published in the literature and 50 At this point, the sequences are examined to identify poten sequences which we have determined ourselves using pro tial probe regions. Two important objectives in designing a cedures known in the art. We use a Sun Microsystems (TM) probe are to maximize homology to the target sequence(s) computer for comparative analysis. The compiler is capable (greater than 90% homology is recommended) and to mini of manipulating many sequences of data at the same time. mize homology to non-target sequence(s) (less than 90% Computers of this type and computer programs which may 55 homology to nontargets is recommended). We have identi be used or adapted for the purposes herein disclosed are fied the following useful guidelines for designing probes commercially available. with desired characteristics. Generally, only a few regions are useful for distinguishing First, probes should be positioned so as to minimize the between closely related species of a phylogenetically con stability of the probe:nontarget nucleic acid hybrid. This served genus, for example, the region 400-500 bases from 60 may be accomplished by minimizing the length of perfect the 5' end of the 16S rRNA molecule. An analysis of closely complementarity to non-target organisms, avoiding G and C related organisms (FIGS. 6, 7 and 8) reveals the specific rich regions of homology to non-target sequences, and by positions (variable regions) which vary between closely positioning the probe to span as many destabalizing mis related organisms. These variable regions of rRNA mol matches as possible (for example, dG:rU base pairs are less ecules are the likely candidates for probe design. 65 destabalizing than some others). FIGS. 5, 6 and 7 display the variations in 16S and 23S Second, the stability of the probe: target nucleic acid rRNA's between two different bacteria with decreasing hybrid should be maximized. This may be accomplished by 5,547,842 7 8 avoiding long A and Trich sequences, by terminating the synthesis using cyanoethylphosphoramidite precursors. Bar hybrids with G:C base pairs and by designing the probe with one, A. D. et al., Nucleic Acids Research 12, 4051-4060 an appropriate Tm. The beginning and end points of the (1984). In this method, deoxyoligonucleotides are synthe probe should be chosen so that the length and % G and % sized on solid polymer supports. Release of the oligonucle Cresult in a Tm about 2-10° C. higher than the temperature at which the final assay will be performed. The importance otide from the support is accomplished by treatment with and effect of various assay conditions will be explained ammonium hydroxide at 60° C. for 16 hours. The solution further herein. Third, regions of the rRNA which are known is dried and the crude product is dissolved in water and to form strong structures inhibitory to hybridization are less separated on polyacrylamide gels which generally may vary preferred. Finally, probes with extensive self-complementa from 10–20% depending upon the length of the fragment. rity should be avoided. 10 The major band, which is visualized by ultraviolet back In some cases, there may be several sequences from a lighting, is cut from the gel with a razor blade and extracted particular region which will yield probes with the desired with 0.1M ammonium acetate, pH 7.0, at room temperature hybridization characteristics. In other cases, one sequence for 8-12 hours. Following centrifugation, the supernatant is may be significantly better than another which differs merely filtered through a 0.4 micron filter and desalted on a P-10 by a single base. 15 column (Pharmacia). Other well known methods for con The following chart indicates how, for one embodiment of struction of synthetic oligonucelotides may, of course, be the invention useful in the detection of a nucleic acid in the employed. presence of closely related nucleic acid sequences, unique Current DNA synthesizers can produce large amounts of sequences can be selected. In this example, rRNA sequences synthetic DNA. After synthesis, the size of the newly made have been determined for organisms A-E and their 20 DNA is examined by gel filtration and molecules of varying sequences, represented numerically, are aligned as shown. It size are generally detected. Some of these molecules repre is seen that sequence 1 is common to all organisms A-E. sent abortive synthesis events which occur during the syn Sequences 2-6 are found only in organisms A, B and C, thesis process. As part of post-synthesis purification, the while sequences 8, 9 and 10 are unique to organism A. synthetic DNA is usually size fractionated and only those Therefore, a probe complementary to sequences 8, 9 or 10 25 molecules which are the proper length are kept. Thus, it is would specifically hybridize to organism A. possible to obtain a population of synthetic DNA molecules of uniform size. Illustrative Pattern of Sequence It has been generally assumed, however, that synthetic Relationships Among Related Bacteria DNA is inherently composed of a uniform population of 30 molecules all of the same size and base sequence, and that Organism rRNA Sequence the hybridization characteristics of every molecule in the A 1 2 3 4 5 6 7 8 9 10 preparation should be the same. In reality, preparations of B 1 2 3 4 5 6 7 11 12 13 synthetic DNA molecules are heterogeneous and are com C 1 2 3 4 5 6 14 15 1617 posed of significant numbers of molecules which, although D 1 18 19 20 21 22 23 24 25 26 35 the same size, are in some way different from each other and E 1 18 19 20 21 27 28 29 30 31 have different hybridization characteristics. Even different preparations of the same sequence can sometimes have In cases where the patterns of variation of a macromol different hybridization characteristics. ecule are known, for example, rRNA, one can focus on Accordingly, preparations of the same synthetic probe specific regions as likely candidates for probe design. How 40 sequence can have different hybridization chacteristics. ever, it is not always necessary to determine the entire Because of this the specificity of probe molecules from nucleic acid sequence in order to obtain a probe sequence. different preparations can be different. The hybridization Extension from any single oligonucleotide primer can yield characteristics of each preparation should be examined in up to 300-400 bases of sequence. When a single primer is order to determine the hybridization cenditions which must used to partially sequence the rRNA of the target organism 45 be used in order to obtain the desired probe specificity. For and organisms closely related to the target, an alignment can example, the synthetic probe described in Example 4 below be made as outlined above. Plainly, if a useful probe has the specificity profile described in Table 14. This data sequence is found, it is not necessary to continue rRNA was obtained by using the hybridization and assay condi sequencing using other primers. If, on the other hand, no tions described. A separate preparation of this probe which useful probe sequence is obtained from sequencing with a 50 has different hybridization characteristics may not have first primer, or if higher sensitivity is desired, other primers precisely the same specificity profile when assayed under the can be used to obtain more sequences. In those cases where conditions presented in Example 4. Such probe preparations patterns of variation for a molecule are not well understood, have been made. To obtain the desired specificity, these more sequence data may be required prior to probe design. probes can be hybridized and assayed under different con Thus, in Examples 1-3 below, two 16S-derived primers 55 ditions, including salt concentration and/or temperature. The were used. The first primer did not yield probe sequences actual conditions under which the probe is to be used must which met the criteria listed herein. The second primer be determined, or matched to extant requirements, for each yielded probe sequences which were determined to be useful batch of probe since the art of DNA synthesis is somewhat following characterization and testing for specificity as imperfect. described. In Example 4, six 23S primers were used prior to 60 Following synthesis and purification of a particular oli locating the probe sequence set forth. gonucleotide sequence, several procedures may be utilized Once a presumptive unique sequence has been identified, to determine the acceptability of the final product. The first a complementary DNA oligonucleotide is synthesized. This is polyacrylamide gel electrophoresis, which is used to single stranded oligonucleotide will serve as the probe in the determine size. The oligonacleotide is labelled using, for DNA/rRNA assay hybridization reaction. Defined oligo 65 example, 'P-ATP and T. polynucleotide kinase. The nucleotides may be synthesized by any of several well labelled probe is precipitated in ethanol, centrifuged and the known methods, including automated solid-phase chemical dried pellet resuspended in loading buffer (80% formamide, 5,547,842 9 10 20 mM NaOH, 1 mM EDTA, 0.1% bromophenol blue and We have discovered that the length of the target nucleic 0.1% xylene cyanol). The samples are heated for five acid sequence and, accordingly, the length of the probe minutes at 90° C. and loaded onto a denaturing polyacryla sequence can also be important. While it is possible for mide gel. Electrophoresis is carried out in TBE buffer (0.1M nucleic acids that are not perfectly complementary to Tris HCl pH 8.3, 0.08M boric acid, 0.002M EDTA) for 1-2 hybridize, the longest stretch of perfectly homologous base hours at 1,000 volts. Following electrophoresis of the oli sequence will normally primarily determine hybrid stability. gonucleotide the gel is exposed to X-ray film. The size of the oligonucleotide is then computed from the migration of While oligonucleotide probes of different lengths and base oligonucleotide standards run concurrently. composition may be used, oligonucleotide probes preferred The sequence of the synthetic oligonucleotide may also be in this invention are between about 15 and about 50 bases in checked by labelling it at the 5' end with P-ATP and T 10 length and are at least about 75-100% homologous to the polynucleotide kinase, subjecting it to standard chemical target nucleic acid. For most applications 95-100% homol degradation techniques, Maxam, A. M. and Gilbert, W., ogy to the target nucleic acid is preferred. Proc. Natl. Acad. Sci., USA 74, 560-564 (1980), and ana Ionic strength and incubation temperature should also be lyzing the products on polyacrylamide gels. Preferably, the taken into account in constructing a probe. It is known that nucleotide sequence of the probe is perfectly complementary 15 the rate of hybridization will increase as ionic strength of the to the previously identified unique rRNA sequence, although reaction mixture increases and that the thermal stability of it need not be. hybrids will increase with increasing ionic strength. In The melting profile, including the melting temperature general, optimal hybridization for synthetic oligonucleotide (Tm) of the oligonucleotide?rRNA hybrids should also be probes of about 15-50 bases in length occurs approximately determined. One way to determine Tm is to hybridize a 20 5° C. below the melting temperature for a given duplex. 'P-labelled oligonucleotide to its complementary target Incubation at temperatures below the optimum may allow nucleic acid at 50° C. in 0.1M phosphate buffer, pH 6.8. The mismatched base sequences to hybridize and can therefore hybridization mixture is diluted and passed over a 2 cm result in reduced specificity. hydroxyapatite column at 50° C. The column is washed with As to nucleic acid concentration, it is known that the rate 0.1M phosphate buffer, 0.02% SDS to elute all unhybrid 25 of hybridization is proportional to the concentration of the ized, single-stranded probes. The column temperature is two interacting nucleic acid species. Thus, the presence of then dropped 15° C. and increased in 5°C. increments until compounds such as dextran and dextran sulphate are thought all of the probe is single-stranded. At each temperature, to increase the local concentration of nucleic acid species unhybridized probe is eluted and the counts per minute and thereby result in an increased rate of hybridization. (cpm) in each fraction determined. The number of cpm 30 Other agents which will result in increased rates of hybrid shown to be bound to the hydroxyapatite divided by the total ization are specified in U.S. application Ser. No. 627,795, cpm added to the column equals the percent hybridization of entitled "Accelerated Nueleic Acid Reassociation Method', the probe to the target nucleic acid. filed Jul. 5, 1984, Continuation-in-Part thereof, Ser. No. (net An alternate method for determining thermal stability of yet assigned), filed Jun. 4, 1987, and U.S. application Ser. a hybrid is outlined below. An aliquot of hybrid nucleic acid 35 No. 816,711, entitled "Accelerated Nucleic Acid Reassocia is diluted into 1 ml of either 0.12M phosphate buffer, 0.2% tion Method', filed Jan. 7, 1986, both of which are incor SDS, 1 mM EDTA, 1 mM EGTA or an appropriate hybrid porated by reference. On the other hand, chemical reagents ization buffer. Heat this 1 ml of solution to 45 degrees C. for which disrupt hydrogen bonds such as formamide, urea, 5 minutes and place it into a room temperature water bath to DMSO, and alcohols will increase the stringency of hybrid cool for 5 minutes. Assay this 1 ml of hybrid containing 40 ization. solution over a hydroxyapatite column, capturing the hybrid Selected oligonucleotide probes may be labelled by any of and washing away unbound probe. If a hybridization solu several well known methods. Useful labels include radio tion other than the 0.12M phosphate buffer is used, then a isotopes as well as non-radioactive reporting groups. Isoto dilution of the hybridization solution into the 0.12M phos pic labels include H, S, *p, 'I, Cobalt and 'C. Most phate buffer will be necessary for binding. Keep taking 45 methods of isotopic labelling involve the use of enzymes aliquots of hybrid and diluting into 1 ml of hybridization and include the known methods of nick translation, end solution or into the standard 0.12M phosphate buffer solu labelling, second strand synthesis, and reverse transcription. tion described above while raising the heating temperature 5 When using radio-labelled probes, hybridization can be degrees C. at a time. Continue this until all of the hybrid is detected by autoradiography, scintillation counting, or dissociated. The point where one half of the hybrid is 50 gamma counting. The detection method selected will depend converted to the dissociated form is considered the Tm. The upon the hybridization conditions and the particular radio Tm for a given hybrid will vary depending on the hybrid isotope used for labelling. ization solution being used because the thermal stability Non-isotopic materials can also be used for labelling, and depends upon the concentration of different salts, detergents, may be introduced by the incorporation of modified nucle and other solutes which effect relative hybrid stability during 55 otides through the use of enzymes or by chemical modifi thermal denaturation. cation of the probe, for example, by the use of non Because the extent and specificity of hybridization reac nucleotide linker groups. Non-isotopic labels include tions such as those described herein are affected by a number fluorescent molecules, chemiluminescent molecules, of factors, manipulation of one or more of those factors will enzymes, cofactors, enzyme substrates, haptens or other determine the exact sensitivity and specificity of a particular 60 ligands. We currently prefer to use acridinium esters. probe, whether perfectly complementary to its target or not. In one embodiment of the DNA/rRNA hybridization For example, the base composition of the probe may be assay invention, a labelled probe and bacterial target nucleic significant because G-C base pairs exhibit greater thermal acids are reacted in solution. rRNA may be released from stability as compared to A-T base pairs due to additional bacterial cells by the sonic disruption method described in hydrogen bonding. Thus, hybridization involving comple 65 Murphy, K. A. et al., U.S. application Ser. No. 841,860, mentary nucleic acids of higher G-C content will be stable entitled "Method for Releasing RNA and DNA From Cells', at higher temperatures. filed Mar. 20, 1986, which is incorporated herein by refer 5,547,842 11 12 ence. Other known methods for disrupting cells include the culosis species, between Mycobacterium intracellulare and use of enzymes, osmotic shock, chemical treatment, and non-tuberculosis species, or between Mycobacterium tuber vortexing with glass beads. Following or concurrent with the culosis-complex bacilli and non-tuberculosis species. For release of rRNA, labelled probe may be added in the accurate identification of the infecting Mycobacterial spe presence of accelerating agents and incubated at the optimal cies the clinician must rely on culture results which can hybridization temperature for a period of time necessary to require anywhere from 3 to 8 weeks of growth followed by achieve significant reaction. Following this incubation extensive biochemical testing. Other tests have been devel period, hydroxyapatite may be added to the reaction mixture oped based on the detection of metabolic products from to separate the probe/rRNA hybrids from the non-hybridized Mycobacterium using carbon-14 labelled substrates. In par probe molecules. The hydroxyapatite pellet is washed, 10 ticular, the Bactec (TM) instrument can detect the presence recentrifuged and hybrids detected by means according to of Mycobacterium within 6 to 10 days of the time of the label used. innoculation. Gill, V. J., supra. However, the test does not Twenty-one embodiments illustrative of the claimed distinguish Mycobacterium species. It is often important to inventions are set forth below, in which a synthetic probe or make this determination so that particular drugs to which the probes complementary to a unique rRNA sequence from a 15 organism is susceptible may be prescribed. For traditional target organism, or group of organisms is determined, con culture methods, this requires an additional 2 to 3 weeks and structed and used in a hybridization assay. for the Bactec method, an additional 6 to 10 days. In addition, specific embodiments for Mycoplasma pneu DESCRIPTION OF PARTICULAR moniae, the Mycobacterium, Legionella, Salmonella, EMBODIMENTS 20 Chlamydia trachomatis, Campylobacter, Proteus mirabilis, Mycobacterium are acid-fast, alcohol fast, aerobic, non Enterococcus, Enterobacter cloacae, E. coli, Pseudomonas mobile bacilli. Their lipid content is high and their growth Group I, bacteria, fungi and Neisseria gonorrhoeae are set slow. Mycobacterium avium and Mycobacterium intracel forth in the following examples. lulare are together referred to as M. avium-intracellulare are As indicated by the below examples, the present invention because they are so difficult to differentiate. Recently, the M. 25 has significant advantages over each of these prior art avium complex, which includes M. intracellulare, was methods not only in the enhanced accuracy, specificity and shown to be the second most commonly isolated, clinically simplicity of the test, but also in greatly reducing the time to significant Mycobacterium. Good, R. C. et al., J. Infect, Dis. achieve a diagnosis. The invention makes possible a defini 146, 829-833 (1982). More recent evidence indicates that tive diagnosis and initiation of effective treatment on the these organisms are a common cause of opportunistic infec 30 same day as testing. tion in patients With AIDS (acquired immune deficiency syndrome). Gill, V. J. et al., J. Clin, Microbio. 22, 543-546 (1985). Treatment of such infections in AIDS patients is EXAMPLE 1. difficult because these organisms are resistant to most anti 35 Described below is the preparation of a single strand tuberculosis drugs. Often a combination of five drugs are deoxyoligonucleotide of unique sequence and defined length used in therapy. The severity of these infections also requires which is labelled and used as a probe in a solution hybrid rapid diagnosis which, prior to the invention herein, was not ization assay to detect the presence of rRNA from Myco available. bacterium avium. This unique sequence is specific for the Members of the Mycobacterium tuberculosis complex rRNA of Mycobacterium avium and does not significantly (Mtb) include Mycobacterium tuberculosis, Mycobacterium cross-react under the hybridization conditions of this bobis, Mycobacterium africanum and Mycobacterium Example, with nucleic acids from any other bacterial species microti. The first three are pathogenic for humans while the or respiratory infectious agent, including the closely-related last is an animal pathogen. These organisms produce slowly Mycobacterium intracellulare. This probe is able to distin developing granulomas on the skin or they may invade 45 guish the two species, notwithstanding an approximate 98% internal organs. Tuberculosis of the lungs can be dissemi rRNA homology between the two species. In this Example, nated to other parts of the body by the circulatory system, the as well as in Examples 2 and 3, sequences for M. avium, M. lymph system, or the intestinal tract. Despite advances in tuberculosis complex, M. intracellulare and related organ public health and the advent of effective chemotherapy, isms were obtained by using a specific primer to a highly Mycobacterial disease, tuberculosis in particular, continues 50 conserved region in the 16S rRNA. The sequence of this to represent a major world-wide health problem. primer, derived from E. coli rRNA, was 5'-GGC CGT TAC The classical method for detecting bacteria in a test CCCACCTACTAGCTAAT3'.5 nanograms of primer was sample involves culturing of the sample in order to expand mixed with 1 microgram of each rRNA to be sequenced in the number of bacterial cells present into observable colony the presence of 0.1M KCl and 20 mM Tris-HCl pH 8.3 in a growths which can be identified and enumerated. If desired, 55 final volume of 10 microliters. The reactions were heated 10 the cultures can also be subjected to additional testing in min. at 45° C. and then placed on ice. 2.5 microliters of 'S order to determine antimicrobial susceptibility. Currently, dATP and 0.5 microliters of reverse transcriptase were the most widely used procedures for the detection, isolation added. The sample was aliquoted into 4 tubes, each tube and identification of Mycobacterium species are the acid containing either dideoxy A, G, T, or C. The concentrations fast bacilli (AFB) smear (using either the Ziehl-Neelsen or 60 of these nucleotides are set forth in Lane et al., supra. The fluorochrome techniques), culture methods using Lowen samples were incubated at 40° C. for 30 minutes, and were stein-Jensen media and Middlebrook media, and biochemi then precipitated in ethanol, centrifuged and the pellets cal tests. The AFB relies on the high lipid content of lypholized dry. Pellets were resuspended in 10 microliters Mycobacterium to retain dye after exposure to acidalcohol. formamide dyes (100% formamide, 0.1% bromphenol blue While the AFB smear test is relatively rapid and simple to 65 and 0.1% xylene cyanol), and loaded onto 80 cm 8% performit does not always detect Mycobacteria and will not polyacrylamide gels. The gels were run at 2000 volts for 2-4 differenigate between Mycobacterium avium and non-tuber hours. 5,547,842 13 14 Thus, nucleotide sequences for the 16S rRNA of Myco bacterium avium and what were considered to be its closest TABLE 1 phylogenetic neighbors, Mycobacterium intracellulare and HYBRIDIZATION OF THE M.AWIUMPROBE Mycobacterium tuberculosis, were determined by the TO HOMOLOGOUSTARGET rRNA method of Lane, D. J. et al., Proc. Nat. Acad, Sci. USA plus rRNA minus rRNA 82:6955 (1985). In addition to determining the rRNA sequences for the organisms noted above, a spectrum of M. avium probe 85-95% 0.5% clinically significant Mycobacterium were also sequenced. ck idization cpm bound to hydroxyapatite These included M. fortuitum, M. scrofulaceum and M. 10 % Hybridization total cpm added to reaction chelonae. Selected members of several genera closely Specificity of the probe for M. avium was tested by related to Mycobacterium were also sequenced, including mixing the 'p labeled probe with rRNA released from cells Rhodococcus bronchialis, Corynebacterium xerosis and of 29 other species of mycobacteria by the sonic disruption Nocardia asteroides. techniques described in Murphy et al., U.S. application Ser. 15 No. 841,860. 1x10 cells were suspended in 0.1 ml 5% SDS Partial rRNA sequences from the above organisms were and sonicated for 10 minutes at 50-60° C. 1.0 ml of aligned for maximum nucleotide homology, using commer hybridization buffer (45% sodium diisobutyl sulfosuccinate, cially available software from Intelligenetics, Inc., 1975 El 40 mM phosphate buffer pH 6.8 and 1 mM EDTA) was Camino Real West, Mountain View, Calif. 94040-2216 added and the mixture incubated for 60 minutes at 72 C. (IFIND Program). From this alignment, regions of sequence 20 Following incubation, 4.0 ml of hydroxyapatite solution (0.14M sodium phosphate buffer, pH 6.8, 0.02% SDS and unique to Mycobacterium avium were determined. The 1.0 gram hydroxyapatite per 50 mls solution) was added and probe was selected so that it was perfectly complementary to incubated for 5 minutes at 72° C. The sample was centri a target nucleic acid sequence and so that it had a 10% or fuged and the supernatant removed. 4.0 ml wash solution greater mismatch with the aligned rRNA from its known (0.14M sodium phosphate pH 6.8) was added and sample closest phylogenetic neighbor. A sequence 38 bases in length was vortexed, centrifuged and the supernatant removed. The was chosen. The number of mismatched bases relative to the radioactivity bound to the hydroxyapatite was determined by scintillation counting. The results are shown in Table 2 and Mycobacterium avium sequence were as follows: Mycobac indicate that the probe is specific for Mycobacterium avium terium tuberculosis (8); Mycobacterium intracelluare (5); 30 and does not react with any other mycobacterial species, Mycobacterium scrofulaceum (6); Mycobacterium chelonae including Mycobacterium intracellulare. (12); and Mycobacterium fortuitum (10). The following cDNA sequence was characterized by the TABLE 2 criteria of length, Tm, and sequence analysis as described at 35 HYBRIDIZATION OF THE M.AVIUM pages 7-8 above and was determined to be specific for the PROBE TO MYCOBACTERIAL SPECIES rRNA Mycobacterium avium: Organism ATCC# 2. Probe Bound ACCGCAAAAGCTTTCCACCAGAAGACAT. Mycobacterium africanum 25420 1.0 GCGTCTTGAG. M. asiaticum 25276 1.2 40 M. avium 25291 87.6 This sequence is complementary to a unique segment found M. bovis 19210 1.2 in the 16S rRNA of Mycobacterium avium. The size of the M. bovis (BCG) 19015 1.0 probe is 38 bases. The probe has a Tm of 74° C. and M. chelonae 4472 0.9 M. flavescens 14474 0.9 sequence analysis by the method of Maxam & Gilbert M. fortuitum 6841 1.0 (1980), supra, confirmed that the probe was correctly syn 45 M. gastri 15754 1.2 M. gordonae 14470 1.2 thesized. The probe is capable of hybridizing to rRNA of M. M. haemophilum 2.9548 13 avium in the region corresponding to bases 185-225 of E. M. intracallulare 3950 1.5 coli 16S rRNA. M. kansasii 12478 1.2 To demonstrate the reactivity of this sequence for Myco M. malmoense 29571 1.2 50 M. marinum 827 1.2 bacterium avium, it was tested as a probe in hybridization M. nonchromogenicum 1930 1.1 reactions under the following conditions. 'P-end-labeled M. phlei 11758 1.3 M. scrofulaceum 19981 1.2 oligonucleotide probes were mixed with 1 microgram M. shinoidei 27962 2.3 (7x10 moles) of purified rRNA from Mycobacterium M. siniae 25275 1.2 M. smegmatis el4468 1.0 avium and reacted in 0.12M PB hybridization buffer 55 M. Szulgai 23069 1.0 (equimolar amounts of Na2HPO and NaH2PO), 1 mM M. terrae 15755 1.2 EDTA and 0.02% SDS (sodium dodecyl sulfate) at 65° C. M. thermoresistibile 19527 1.3 for 60 minutes in a final volume of 50 microliters. In M. trivials 23292 1.2 M. tuberculosis (avirulent) 25177 1.4 separate tubes the probe was mixed with the hybridization M. tuberculosis (virulent) 27294 1.1 buffer both with and without target present. Following 60 M. ulcerans 19423 1.4 M. vaccae 15483 1.2 separation on hydroxyapatite as outlined in the patent appli M. cenopi 1997 15 cations identified at page 2, supra, the hybrids were quan titated by scintillation counting. These results are presented As shown in Table 3 the probe also did not react with the in Table 1, showing that the probe has a high extent of 65 rRNA from any of the respiratory pathogens which were reaction to homologous target and very little non-specific also tested by the method just described. Nor did the probe binding to the hydroxyapatite. react with any other closely related or phylogenetically more 5,547,842 15 16 diverse species of bacteria also tested by that method (Table in a final volume of 50 microliters. In separate tubes the 4). probe was mixed with the hybridization buffer with and without target Mycobacterium intracellulare rRNA present. TABLE 3 Following separation on hydroxyapatite as outlined previ HYBRDIZATION OF M. AVIUMPROBETO ously the hybrids were quantitated by scintillation counting. RESPIRATORY PATHOGENS These results are shown in Table 5. Organism ATCC % Probe Bound TABLE 5 Corynebacterium xerosis 373 0.7 HYBRDIZATION OF THE M. INTRACELLULARE Fusobacterium nucleatum 25586 1.3 10 Haemophilum influenzae 19418 1.3 PROBE TO HOMOLOGOUSTARGET rRNA*/ Klebsiella pneumoniae 23357 1.8 Legionella pneumophila 33152 0.0 plus rRNA minus rRNA Mycoplasma pneumoniae 15531 3.0 Neisseria meningitidis 13090 0.0 M. intracellulare probe 85-95% 0.5% Pseudomonas aeruginosa 25330 0.0 15 sk idization-Cpm bound to hydroxyapatite Propionibacterium acnes 6919 11 % Hybridization total cpm added to reaction Streptococcus pneumoniae 6306 0.0 Staphylococcus aureus 25923 1.5 These data shows that the probe has a high extent of reaction to its homologous target and very little non-specific TABLE 4 20 binding to the hydroxyapatite. Specificity of the Mycobacterium intracellulare probe HYBRIDIZATION OF THE M.AVIUMPROBE TO APHY was tested by mixing the 'P labelled probe with rRNA LOGENETIC CROSS SECTION OF BACTERIAL SPECIES released from cells from 29 other species of mycobacteria by Organism ATCC# 96 Probe Bound sonic disruption techniques described in Murphy et. al. U.S. 25 Pat. application Ser. No. 841,860. All hybridization assays Acinetobacter calcoaceticus 33604 0.0 Branhamella catarrahalis 25238 0.6 were carried out as described in Example 1. Table 6 indicates Bacilius subtilis 6051 0.9 that the probe is specific for Mycobacterium intracellulare Bacteroides fragilis 23745 1.0 and does not react with any other mycobacterial species, Campylobacter jejuni 33560 0.4 including Mycobacterium avium. These results are impres Chromobacterium violaceum 29094 1.7 30 Clostridium perfringens 13124 2.1 sive in view of the 98% rRNA homology to M. avium, 98% Deinococcus radiodurans 35073 0.8 homology to M. kansasii; 98% homology to M. asiaticum, Deria gummosa 15994 0.3 and 97% homology to M. tuberculosis. Enterobacter aerogenes 13048 0.6 Escherichia coli 11775 0.3 TABLE 6 Mycobacterium gordonae 14470 1.9 35 Mycoplasma hominis 14027 3.3 HYBRIDIZATION OF THEM, INTRACELLULARE PROBE Proteus mirabilis 2.9906 0.0 TO MYCOBACTERIAL SPECIES Psudomonas cepacia 11762 1.0 Rahnella aquatilis 33071 2.1 Organism ATCCi 9%. Probe Bound Rhodospirillum rubrum 1170 0.6 Streptococcus mitis 9811 0.9 Mycobacterium africanum 25420 0.9 Vibrio parahaemolyticus 17802 1.2 M. asiaticum 25276 1.1 Yersinia enterocolitica 9610 0.4 M. avium 25291 1.3 M. bovis 19210 1.1 M. bovis (BCG) 19015 1.2 M. chelonae 14472 1.0 M. favescens 14474 1.2 EXAMPLE 2 45 M. fortuitum 6841 1.3 M. gastri 15754 13 After the alignment described in Example 1, the following M. gordonae 14470 1.3 sequence was characterized by the aforementioned criteria M. haemopilum 29548 0.9 M. intracellulare 13950 78.8 of length, Tm and sequence analysis and was determined to M. kansasii 12479 1.1 be specific for Mycobacterium intracellulare: 50 M. maimoense 29571 1.0 ACCGCAAAAGCTTTCCACCTAAAGACAT. M. marinum 827 0.9 GCGCCTAAAG M. nonchromogenicum 1930 1.0 M. phlei 1758 1.1 The sequence is complementary to a unique segment found M. scrofulaceum 19981 1.0 in the 16S rRNA of Mycobacterium intracellulare. The size M. shinoidei 27962 3 of the probe was 38 bases. The probe has a Tm of 75° C. and 55 M. simiae 25275 1.1 M. smegmatis e14468 1.3 sequence analysis confirmed, that the probe was correctly M. Szulgai 23069 1.0 synthesized. The probe hybridizes to RNA of M. intracel M. terrae 15755 1.4 lulare in the region corresponding to bases 185-225 of E. M. thermoresistibile 19527 1.6 Colli 16S rRNA. M. triviale 23292 13 60 M. tuberculosis (avirulent) 2577 1.2 To demonstrate the reactivity of this sequence for the M. tuberculosis (virulent) 27294 1.2 Mycobacterium intracellulare, the probe was tested in M. ulcerans 19423 1.1 hybridization reactions under the following conditions. 'P- M. vaccae 15483 1.0 end-labelled oligonucleotide probe was mixed with 1 micro M. xenopi 19971 1.2 gram (7x10' moles) of purified rRNA from Mycobacte rium intracellulare and reacted in 0.12M PB (equimolar 65 As shown in Table 7 the probe did not react with the rRNA amounts of NaHPO, and NaHPO), 1 mM EDTA and from any of the respiratory pathogens tested in the hybrid 0.2% SDS (sodium dodecyl sulfate) at 65° C. for 60 minutes ization assay. Nor did the probe react with any other closely 5,547,842 17 18 related or phylogenetically more diverse species of bacteria SDS (sodium dodecyl sulfate) at 65° C. for 60 minutes in a that were tested (Table 8). final volume of 50 microliters. In separate tubes the probe was mixed with the hybridization buffer with and without TABLE 7 target rRNA from Mycobacterium tuberculosis present. Fol HYBRIDIZATION OF THEM.INTRACELLULARE PROBE lowing separation on hydroxyapatite as outlined previously TO RESPRATORY PATHOGENS the hybrids were quantitated by scintillation counting. The Organism ATCC 76 Probe Bound results are shown in Table 9. Corynebacterium xerosis 373 2.2 TABLE 9 Fusobacterium nucleatum 25586 1.5 10 Haemophilum influenzae 19418 1.3 HYBRIDIZATION OF ME-COMPLEX 16S rRNA DNA Klebsiella pneumoniae 23357 1.2 PROBE TO HOMOLOGOUSTARGET rRNA*f Legionella pneumophia 3352 1.2 Mycoplasma pneumoniae 15531 3.2 plus rRNA minus rRNA Neisseria meningitidis 13090 1. Pseudomonas aeruginosa 25330 10 5 Mtb complex probe 85-95% 0.5% Propionibacterium acnes 6919 2.9 Streptococcus pneumoniae 6306 1.6 s idization cpm bound to hydroxyapatite Staphylococcus aureus 25923 1.3 % Hybridization total cpm added to reaction This data shows that the probe has a high extent of TABLE 8 reaction to homologous target and very little non-specific binding to the hydroxyapatite. HYBRIDIZATION OF THEM.INTRACELLULARE PROBE TO APHYLOGENETIC CROSS SECTION OF Specificity of the probe for the Mtb complex was tested by BACTERIAL SPECIES mixing the 'plabelled probe with rRNA released from cells of the 4Mtb complex bacilli and of 25 other mycobacterial Organism ATTC a Probe 25 species by sonic disruption techniques described in Murphy Acinetobacter calcoaceticus 33604 1.5 et. al., U.S. patent application Ser. No. 841,860. All hybrid Branhamella catarrhais 25238 8 ization assays were carried out as described in Example 1. Bacilius subtilis 605. 1.7 Bacteroides fragiles 23745 1.9 Table 10 indicates that the probe is specific for organisms Campylobacter jejuni 33560 9 30 within the Mtb complex and does not react with any other Chromobacterium violaceum 29094 1.4 mycobacterial species. Clostridium perfringens 13124 2.1 Deinococcus radiodurans 35073 2.1 TABLE 10 Deria gummosa 15994 1.6 Enterobacter aerogenes 13048 1.3 HYBRDIZATION OF Mtb-COMPLEX 16S rRNA DNA Escherichia coli 1775 1.2 35 PROBE TO MYCOBACTERIAL SPECIES Mycobacterium gordonae 14470 2.3 Mycoplasma hominis 1402.7 2.6 Organism ATCC 6 Probe Bound Proteus mirabilis 2.9906 1.2 Pseudomonas cepacia 1762 1.7 Mycobacterium africanum 25420 68. Rahnella aquatilis 33071 1.5 M. asiaticum 25276 3.4 Rhodospiriliura rubrum 1170 1.4 40 M. avium 25291 0.9 Striptococcus mitis 98.1 14 M. bovis 19210 63.1 Vibrio parahaemolyticus 17802 2.5 M. chelonae 14472 .1 ersinia enterocolitica 960 1.1 M. flavescens 14474 0.9 M. fortuitum 6841 1.1 M. gastri 15754 0.8 M. gordonae 14470 1.1 EXAMPLE 3 45 M. haemophilum 29548 0.8 M. intracallulare 3950 1.1 After the alignment described in Example 1, the following M. kansasii 12479 3 sequence was characterized by the aforementioned three M. maimoense 2957 0.9 M. marinum 827 1. criteria of size, sequence and Tm, and was determined to be M. nonchromogenicum 1930 1. specific to the Mtb complex of organisms, Mycobacterium 50 M. phlei 11758 13 tuberculosis, Mycobacterium, africanum, Mycobacterium M. scrofulaceum 19981 1.1 bovis, and Mycobacterium microti: M. shinoidei 21962 10 M. siniae 252.75 1.2 1. TAAAGCGCTTTCCACCACAAGACATG M. smegmatis e14468 0.9 CATCCCGTG. M. Szulgai 23069 1.1 The sequence is complementary to a unique segment found 55 M. terrae 15755 1.0 M. thermoresistibile 19527 1.0 in the 16S rRNA of the Mtb-complex bacteria. The size of M. triviale 23292 1.2 the probe is 35 bases. The probe has a Tm of 72° C. and M. tuberculosis (avirulent) 25177 66.2 sequence analysis confirmed that the probe was correctly M. tuberculosis (virulent) 27294 62.4 synthesized. It is capable of hybridizing in the region M. ulcerans 19423 0.9 corresponding to bases 185-225 of E. coli 16S rRNA. 60 M. vaccae 15483 0.8 To demonstrate the reactivity of this sequence for the Mtb M. cenopi 19971 2.6 complex the probe was tested in hybridization reactions under the following conditions. 'P-end-labelled oligonucle As shown in Table 11 the probe did not react with the otide probe was mixed with 1 microgram (7x10 moles) of rRNA from any of the respiratory pathogens tested in the purified rRNA from Mycobacterium tuberculosis and 65 hybridization assay. Nor did the probe react with any other reacted in 0.12M PB hybridization buffer (equimolar closely related or phylogenetically more diverse species of amounts of Na2HPO, and NaH2PO), 1 mM EDTA and 0.2 bacteria that were tested (Table 12). 5,547,842 19 20 TABLE 11 TABLE 13-continued HYBRIDIZATION OF Mtb-COMPLEX 16S rRNA DNA HYBRIDIZATION OF PROBE OF EXAMPLES 3 AND 2 PROBE TO RESPERATORY PATHOGENS DERVATIVES THEREOF TO MYCOBACTERIAL SPECIES Organism ATCC % Probe Bound Example Corynebacterium aerosis 373 1.3 % % % Fusobacterium bucleatum 25586 1.0 Probe 1 Probe 2 Probe 3 Haemophilum influenzae 19418 1.6 Organism ATCC Bound Bound Bound Klebsiella pneumoniae 23357 2 10 Legionella pneumophila 33152 1.4 M. bovis 19210 63. 75.3 74 Mycoplasma pneumoniae 15531 1. M. chelonae 14472 1.1 1.5 1.6 Neisseria meningitidis 13090 1.0 M. flavescens 14474 0.9 2.7 1.4 Pseudononas aeruginosa 25330 1.7 M. fortuitum 684.1 1. 3.6 15 Propionibacterium acnes 6919 1.2 M. gastri 15754 0.8 3.6 1.7 Streptococcus pneumoniae 25923 0.9 M. gordonae 14470 1.1 1.6 1.4 15 M. haemophilum 2.9548 0.8 3.2 1.7 M. intracellulare 13950 1.1 1.6 1.4 M. kansasii 12478 1.3 2. 2.0 TABLE 12 M. maimoense 29571 0.9 2.8 15 M. marinum 827 1.1 2.1 1.5 HYBRIDIZATION OF THE Mtb-COMPLEX 16S M. nonchromogenicum 1930 1.1 3.0 15 rRNA DNA PROBE TO APHYLOGENETIC CROSS 20 M. phlei 11758 13 1.3 1.1 SECTION OF BACTERIAL SPECIES M. scrofulaceum 19981 1. 3.4 1.6 M. shinoidei 27962 1.0 2.7 1.6 Organism ATCC % Probe M. simiae 25275 1.2 2.9 1.8 M. smegmatis e14468 0.9 15 1.2 Acinetobacter calcoaceticus 33604 1.3 M. Szulgai 23069 1.1 3.6 1.1 Branhamella catarrhalis 25238 15 25 M. terrae 15755 1.0 3.7 2.0 Bacilius subtilis 605. 1.3 M. thermoresistibile 19527 1.0 1.6 1.3 Bacteroides fragilis 23745 1.3 M. triviale 23292 1.2 1.6 2.0 Campylobacter jejuni 33560 1.1 M. tuberculosis 25177 66.2 75 68 Chromobacterium violaceum 29094. 1.0 (avirulent) Clostridium perfringens 13124 1.2 M. tuberculosis 27294 62.4 74 75 Deinococcus radiodurans 35073 1.0 30 (virulent) Derxia gummosa 15994 1.0 M. ulcerans 19423 0.9 17 3.0 Enterobacter aerogenes 13048 1.0 M. vaccae 15483 0.8 1.4 1.2 Escherichia coli 11775 10 Mycobacterium gordonae 14470 1.3 M. xenopi 1997 2.6 1.4 1.2 Mycoplasma hominis 1402.7 0.5 Proteus mirabilis 299.06 1.0 Pseudomonas cepacia 1762 2.6 35 Rahnella aquatilis 33071 1.9 Rhodospirillum rubrum 11170 0 EXAMPLE 4 Streptococcus mitis 981 1.1 Vibrio parahaemolyticus 17802 0.9 The probe specific for the 23S rRNA of the M tuberculosis Yersinia enterocolitica 96.10 1.1 complex was obtained by using a primer which was comple 40 mentary to a highly conserved region of 23S rRNA. The sequence of this primer, derived from E. coli rRNA, was Two derivatives of the probe of Example 3 (numbered 2-3 5'-AGG AACCCTTGG GCTTTCGG-3'. Five nanograms below) were made and tested: of this primer was mixed with 1 microgram of rRNA from 2. CCGCTAAAGCGCTTTCCACCACAAGA M. tuberculosis and other closely related Mycobacterium CATGCATCCCG 45 and the procedure as described for Examples 1, 2 and 3 was 3. ACACCGCTAAAGCGCTTTCCACCACAA followed. After alignment as described in Example 1, the GACATGCATC. following sequence was determined to be specific to the Mtb All three probes have similar Tms (72; 73.5°; and 72.3°, complex of Organisms, Mycobacterium tuberculosis, Myco respectively) and similar hybridization characteristics bacterium africanum, Mycobacterium bovis, and Mycobac Hybridization to Mycobacterium tuberculosis complex 50 terium microti: organisms was 68–75% and non-specific hybridization to TGCCCTACCCACACCCACCACAAGGTGATGT hydroxyapatite was less than 2%. Results of hybridization The sequence is complementary to a unique segment found assay tests for these derivatives follow. in the 23S rRNA of the Mtb-complex bacteria. The oligo nucleotide probe was characterized as previously described TABLE 13 55 by the criteria of length, Tm and sequence analysis. The size HYBRIDEZATION OF PROBE OF EXAMPLES 3 AND 2 of the probe is 31 bases. The probe has a Tm of 72.5°C. and DERIVATIVES THEREOF sequence analysis confirmed that the probe was correctly TO MYCOBACTERIAL SPECIES synthesized. It is capable of hybridizing in the region corresponding to bases 1155-1190 of E. coli 23S rRNA. Example 60 % % % To demonstrate the reactivity of this sequence for the Mtb Probe Probe 2 Probe 3 complex the probe was tested in hybridization reactions Organism ATCC Bound Bound Bound under the following conditions. 'P-end-labelled oligonucle Mycobacterium 25420 68.1 69.4 70.6 otide probes were mixed with 1 microgram (7x10' moles) africanum of purified rRNA from Mycobacterium tuberculosis and M. asiaticum 25274 3.4 5.3 1.8 65 reacted in 0.12M PB hybridization buffer (equimolar M. avium 25291 0.9 1.6 1.4 amounts of NaHPO, and NaH2PO), 1 mM EDTA and 0.2 SDS (sodium dodecyl sulfate) at 65° C. for 60 minutes in a 5,547,842 21 22 final volume of 50 microliters. In separate tubes the probe The sequence of this Example 5 was obtained using a 23S was mixed with the hybridization buffer with and without primer with the sequence 5'-GGC CAT TAG ATC ACT target rRNA from Mycobacterium tuberculosis present. Fol CC-3'. It was characterized and shown to be specific for the lowing separation on hydroxyapatite as outlined previously Mycobacterium tuberculosis complex of organisms includ the hybrids were quantitated by scintillation counting. The ing Mycobacterium tuberculosis, Mycobacterium africanum results are shown in Table 14. and Mycobacterium bovis. This sequence, from 23S rRNA, TABLE 14 is 31 bases in length and has a Tm of 72°C. This probe is capable of hybridizing to RNA of the aforementioned organ HYBRIDIZATION OF THE Mtb-COMPLEX isms in the region corresponding to bases 540-575 of E. coli 23S rRNA DNA PROBETO 10 HOMOLOGOUSTARGET rRNA 23S rRNA. To demonstrate the reactivity and specificity of this probe plus rRNA minus rRNA for Mycobacterium tuberculosis complex, it was tested as a Mtb complex 23S probc 94% 1.2% probe in hybridization reactions under the following condi 15 tions. 'P-end-labeled oligonucleotide probe was mixed These data show that the probe has a high extent of reaction with rRNA released from cells of 30 species of mycobacteria to homologous target and very little non-specific binding to by the sonic disruption techniques described in Murphy et the hydroxyapatite. al., U.S. patent application Ser. No. 841,860. 3x10" cells Specificity of the probe for the Mtb complex was tested by were suspended in 0.1 ml 5% SDS and sonicated for 15 mixing the 'Plabelled probe with rRNA released from cells 20 minutes at 50°-60° C. One ml of hybridization buffer (45% of the four Mtb complex bacilli and of 25 other mycobac diisobutyl sulfosuccinate, 40 mM phosphate buffer pH 6.8, terial species by sonic disruption techniques described in 1 mM EDTA, 1 mM EGTA) was added and the mixture Murphy et al., U.S. patent application Ser. No. 841,860. All incubated at 72° C. for 2 hours. Following incubation, 4 ml hybridization assays were carried out as described in of 2% (w/v) hydroxyapatite, 0.12M sodium phosphate buffer Example 1. Table 14 indicates that the probe is specific for pH 6.8, 0.02% SDS, 0.02% sodium azide was added and organisms within the Mtb complex and does not react with incubated at 72° C. for 5 minutes. The sample was centri any other mycobacterial species. fuged and the supernatant removed. Four ml wash solution (0.12M sodium phosphate buffer pH6.8, 0.02% SDS, 0.02% TABLE 1.5 sodium azide) was added and the sample was vortexed, 30 centrifuged and the supernatant removed. The radioactivity HYBRIDIZATION OF Mtb-COMPLEX23S rRNA DNA bound to the hydroxyapatite was determined by scintillation PROBE TO MYCOBACTERIAL SPECIES counting. The results are shown in Table 16 and indicate that Organism ATCC is % Probe Bound the probe is specific for the Mycobacterium tuberculosi complex of organisms. Mycobacterium africanum 25420 33.6 35 M. asiaticum 25276 1.2 M. avium 2529 10 TABLE 16 M. bovis 19210 32.0 HYBRIDIZATION OF THE M. TUBERCULOSIS M. chelonae 14472 1.2 COMPLEX PROBE OF EXAMPLE 5TO M. flavescens 14474 1.2 MYCOBACTERIAL SPECIES M. fortuitum 6841 1.3 M. gastri 15754 1.1 40 M. gordonae 14470 1.2 Organism ATCC if % Probe Bound M. haemophilum 2.9548 1.2 Mycobacterium africanum 25420 18.0 M. intracellulare 13950 1.1 M. asiaticum 25274 2.6 M. kansasii 12479 1.3 M. maimoense 29571 1.3 M. avium 25291 3.4 45 M. bovis 19210 21.7 M. marinum 827 1.2 M. bovis (BCG) 35734 35.3 M. nonchromogenicum 1930 1.0 M. chelonae 14472 3.8 M. phlei 11758 1.0 M. flavescens 14474 2.3 M. scrofulaceum 19981 1. M. fortuitum 6841 1.8 M. shinoidei 27962 1.2 M. gastri 15754 2.2 M. simiae 25275 1.3 M. gordonae 14470 2.8 M. smegmatis e14468 1.1 50 M. haemophilum 2.9548 2.8 M. szulgai 23069 1.1 M. intracellulare 13950 2.1 M. terrae 15755 1.0 M. kansasii 12478 1.6 M. thermoresistibile 19527 1.2 M. malmoense 29571 2.3 M. triviale 23292 1.0 M. marinum 827 2. M. tuberculosis (avirulent) 25177 33.7 M. nonchromogenicum 1930 2.3 M. tuberculosis (virulent) 27294 38.1 55 M. phlei 11758 2.1 M. ulcerans 19423 1.3 M. scrofulaceum 19981 2.2 M. vaccae 15483 1.0 M. shimoidei 27962 9 M. xenopi 19971 1.3 M. siniae 25275 2.2 M. smegmatis el4468 20 M. szulgai 23069 2.2 60 M. terrae 15755 2.2 M. thermoresistible 19527 2.2 EXAMPLE 5 M. triviale 23292 2.0 M. tuberculosis (avirulent) 25177 26.4 Three additional Mycobacterium tuberculosis complex M. tuberculosis (virulent) 27294 36.6 probes, Examples 5-7 herein, were identified using two M. ulcerans 19423 2.5 unique primers complementary to 23S rRNA. The first M. vaccae 5483 2.4 sequence is: 65 M. xenopi 19971 2.8 CCATCACCACCCTCCTCCGGAGAGGAAAAGG. 5,547,842 23 24 Table 16 shows that the probe also did not cross react with RNA from any of the closely related organisms tested by the TABLE 18-continued method just described. HYBRIDIZATION OF THE M. TUBERCULOSIS COMPLEX PROBE OF EXAMPLE 6 TOMYCOBACTERIAL SPECIES TABLE 17 Organism ATCC % Probe Bound HYBRDIZATION OF THE M. TUBERCULOSIS COMPLEX PROBE OF EXAMPLE 5TO PHYLOGENETICALLY M. gastri 15754 3.2 CLOSELY RELATED ORGANISMS M. gordonae 14470 3.0 M. haemophilum 2.9548 3.0 Organism ATCC it % Probe Bound 10 M. intracellulare 13950 3.6 M. kansasii 12478 3.9 Actinomadura madurae 1942.5 2.1 M. malnoense 29571 2.9 Actinoplanes italicus 100.49 3.1 M. marinun 827 2.9 Arthrobacter oxidans 14358 2.1 M. nonchromogenicum 1930 4.8 Brevibacterium linens e9172 19 M. phlei 1758 2.9 Corynabacterium xerosis 373 2.2 M. scrofulaceum 19981 2.6 Dermatophilus congolensis 14367 2.2 M. shinoidei 27962 3.6 Microbacterium lacticum 818O 2. M. siniae 25275 3.3 Nocardia asteroides 19247 2.0 M. smegmatis el4468 3.0 Nocardia brasiliensis 19296 2.2 M. Szulgai 23069 2.8 Nocardia otitidis-caviarum 14629 2.0 M. terrae 15755 2.8 Nocardioposis dassonvillei 2328 40 M. thermoresistibile 19527 11.7 Oerskovia turbata 33225 2.2 20 M. triviale 23292 3.2 Oerskovia xanthineolytica 274.02 2.0 M. tuberculosis (avirulent) 25177 65.0 Rhodococcus aichiensis 33611 1.9 M. tuberculosis (virulent) 27294 53.0 Rhodococcus aurantiacus 25938 2.0 M. ulcerans 19423 2.5 Rhodococcus bronchialis 25592 2. M. vaccae 15483 2.8 Rhodococcus chubuensis 33609 2.3 M. cenopi 1997 3.3 Rhodococcus equi 6939 2.4 25 Rhodococcus obuensis 33610 2.2 Rhodococcus sputi 29627 2.3 Table 19 shows that the probe also did not cross react with RNA from any of the phylogenetically closely related organ isms tested by the methodjest described. EXAMPLE 6 30 TABLE 19 The second Mycobacterium tuberculosis complex probe HYBRIDIZATION OF THE M. TUBERCULOSIS COMPLEX was obtained using a 23S primer with the sequence 5' CCT PROBE OF EXAMPLE 6 TO PHYLOGENETICALLY GATTGC CGT CCA GGT TGA GGG AAC CTT TGG CLOSELY RELATED ORGANISMS G-3'. Its sequence is: 35 Organism ATCC % Probe Bound

CTGTCCCTAAACCCGATTCAGGGTTCGAGGTTAGATGC Actinomadura madurae 1942.5 1.3 Actinoplanes italicus 10049 0.6 This sequence, from 23S rRNA, is 38 bases in length and has Arthrobacter oxidans 14358 1.1 aTm of 75°C. It hybridizes in the region corresponding to Brevibacterium linens e972 0.8 40 Corynebacterium erosis 373 1.0 bases 2195-2235 of E. coli 23S rRNA. Dermatophilus congolensis 14367 0.6 Like the complex probe in Example 5, this sequence was Microbacterium lacticum 818O 19 characterized and shown to be specific for the Mycobacte Nocardia asteroides 19247 0.9 rium tuberculosis complex of organisms including Myco Nocardia brasiliensis 19296 0.8 Nocardia otitidis-caviarum 14629 15 bacterium tuberculosis, Mycobacterium africanum and Nocardioposis dassonvillei 2328 0.5 Mycobacterium bovis. 45 Oerskovia turbata 33225 0.3 To demonstrate the reactivity and specificity of the probe Oerskovia xanthineolytica 274.02 0.8 of this Example 6 to Mycobacterium tuberculosis complex, Rhodococcus aichiensis 3361 1.6 Rhodococcus aurantiacus 25938 0.7 it was tested as a probe in hybridization reactions under the Rhodococcus bronchialis 25592 15 following conditions described for the probe in Example 5. Rhodococcus chubuensis 33609 0.8 The results are shown in Table 18 and indicate that the probe 50 Rhodococcus equi 6939 0.3 is specific for the Mycobacterium tuberculosis complex of Rhodococcus obitensis 33610 0.8 organisms with the exception of Mycobacterium thermore Rhodococcus sputi 29627 1.4 sistibile, a rare isolate which is not a human pathogen. TABLE 1.8 55 EXAMPLE 7 HYBRDIZATION OF THE M. TUBERCULOSIS COMPLEX The following additional Mycobacterium tuberculosis PROBE OF EXAMPLE 6 TOMYCOBACTERIAL SPECIES complex probe also has been identified using a 23S primer Organism ATCC is % Probe Bound with the same sequence as that of Example 6, namely, 60 5'-CCT GATTGC CGT CCA GGTTGA GGG AAC CTT Mycobacterium africanum 25420 56.0 TGG G-3': M. asiaticum 25274 3.1 M. avium 25291 2.6 AGGCACTGTCCCTAAACCCGATTCAGGGTTC. M. bovis 19210 48.0 This sequence, from 23S rRNA is 31 bases in length and M. bovis (BCG) 35734 63.0 has a Tm of 71°C. It hybridizes in the region corresponding M. chelonae 14472 2.8 M. flavescens 14474 2.8 65 to bases 2195-2235 of E. coli 23S rRNA. As is the case with M. fortuitum 6841 3.0 the Mycobacterium tuberculosis complex probes of Examples 5 and 6 herein, this sequence also was character 5,547.842 25 26 ized and shown to be specific for the Mycobacterium tuber culosis complex of organisms, including Mycobacterium TABLE 21-continued tuberculosis, Mycobacterium africanum and Mycobacte HYBRIDIZATION OF THE M. TUBERCULOSIS COMPLEX rium bovis. PROBE OF EXAMPLET TO PHYLOGENETICALLY To demonstrate the reactivity and specificity of this probe CLOSELY RELATED ORGANISMS for Mycobacterium tuberculosis complex, it was tested as a probe in hybridization reactions under the conditions Organism ATCC if % Probe Bound described for the probe of Example 5. Table 20 shows that Rhodococcus chubuensis 33609 0.6 the probe is specific for the Mycobacterium tuberculosis Rhodococcus equi 6939 0.6 10 Rhodococcus obuensis 33610 0.6 complex of organisms. Rhodococcus sputi 29627 0.9 TABLE 20 Notably, overlapping probes may have identical specific HYBRIDIZATION OF THE MYCOBACTERIUM ity. Compare, for example, the probes of Examples 6 and 7: TUBERCULOSIS COMPLEX PROBE OF EXAMPLE 7TO MYCOBACTERIAL SPECIES 15 Ex. 6 CTGTCCCTAAACCCGATTCAGGGTTC GAGGTTAGATGC Organism ATCC is % Probe Bould Ex. 7 AGGCACTGTCCCTAAACCCGAT Mycobacterium africanum 25420 43.0 TCAGGGTTC M. asiaticum 25274 0.6 There may be several sequences from a particular region M. avium 25291 0.7 20 which will yield probes with the desired hybridization M. boyis 1920 43.0 M. boyis (BCG) 35734 46.0 characteristics. In other cases, one probe sequence may be M. chelonae 14472 0.6 significantly better than another probe differing by a single M. flavescens 14474 0.6 base. In general, the greater the sequence difference (% M. fortuitum 6841 0.5 mismatch) between a target and nontarget organism, the M. gastri 15754 0.9 25 M. gordonae 14470 0.7 more likely one will be able to alter the probe without M. haemophilum 2.9548 0.6 affecting its usefulness for a specific application. This phe M. intracellulare 13950 0.6 nomenon also was demonstrated by the derivative probes in M. kansasii 12478 0.9 Example 3. M. naimoense 2957 0.8 In Example 7, five bases were added to the 5' end of the M. marinum 827 0.7 30 M. nonchromogenicum 1930 0.8 probe in Example 6, and 12 bases were removed from the 3' M. phlei 11758 0.6 end. The two probes have essentially identical hybridization M. scrofulaceum 19981 0.7 characteristics. M. shimoidei 27962 0.8 M. siniae 25275 0.7 M. smegmatis el4468 0.6 M. szulgai 23069 0.6 35 EXAMPLE 8 M. terrae 15755 0.7 M. thermoresistibile 19527 0.9 The Mycobacterium genus is particularly difficult to dis M. triviale 23292 0.7 M. tuberculosis (avirulent) 25177 40.0 tinguish from Nocardia, Corynebacterium and Rhodococ M. tuberculosis (virulent) 27294 50.0 cus. These genera have common antigens, precipitins and G M. ulcerans 19423 0.17 40 & C counts. Despite the fact that these organisms also M. vaccae 15483 0.4 exhibit 92-94% rRNA homology to the above listed Myco M. xenopi 19971 0.6 bacterium organisms, we have designed probes which detect all members of the genus Mycobacterium without cross Table 21 shows that the probe also did not cross react with reacting to the related genera. RNA from any of the closely related organisms tested by the 45 In addition to the Mycobacterium species probes already method just described. disclosed, four probes specific for members of the Myco bacterium genus were identified using one primer comple TABLE 21 mentary to 16S rRNA and one primer complementary to 23S HYBREDIZATION OF THE M. TUBERCULOSIS COMPLEX rRNA. Sequence 1 was obtained using a 16S primer with the PROBE OF EXAMPLET TO PHYLOGENETICALLY 50 sequence 5'-TTA CTA GCG ATT CCG ACT TCA-3'. CLOSELY RELATED ORGANISMS Sequences 2, 3 and 4 were obtained using a 23S primer with Organism ATCC if % Probe Bound the sequence 5'-GTG TCG GTT TTG GGT ACG-3'. Sequence 1 is capable of hybridizing to RNA of the genus Actinomadura madurae 1942.5 1.0 Mycobacterium in the region corresponding to bases Actinoplanes italicus 10049 0.6 55 Arthrobacter oridans 14358 0.4 1025-1060 of E. coli 16S rRNA. Sequences 2-4 hybridize Brevibacterium linens e9172 0.8 in regions corresponding to the following bases of E. coli Corynebacterium xerosis 373 0.6 23S rRNA in our numbering system (See FIG. 2); Dermatophilus congolensis 14367 0.8 1440-1475; 1515-1555; 1570–1610 in our numbering sys Microbacterium lacticum 8180 0.5 ten. Nocardia asteroides 19247 0.7 60 Nocardia brasiliensis 19296 0.5 The following sequences were characterized and shown to Nocardia otitidis-caviarum 14629 0.6 be specific for the genus Mycobacterium: Nocardioposis dassonvillei 23218 0.6 Oerskovia turbata 33225 0.8 1. CCATGCACCACCTGCACACAG GCCACAAGG Oerskovia xanthineolytica 27402 0.6 2. GGCTTG CCC CAGTATTAC CAC TGA CTG GTA Rhodococcus aichiensis 33611 0.7 CGG Rhodococcus aurantiacus 25938 0.7 65 Rhodococcus bronchialis 25592 0.6 3. CAC CGAATTCGC CTC AAC CGG CTATGC GTC ACC TC 5,547,842 27 28 4. GGG GTACGG CCC GTGTGT GTG CTCGCTAGA EDTA, 1 mM EGTA) was added and the mixture incubated GGC at 72° C. for 2 hours. Following incubation, 2 ml of Sequence 1, from 16S rRNA, is 30 bases in length and has separation solution (containing 2.5 g/l cationic magnetic a Tm of 73°. Sequence 2, from 23S rRNA, is 33 bases in microspheres, 0.17M sodium phosphate buffer pH 6.8, 7.5% length and has a Tm of 75° C. Sequence 3, from 23S rRNA, Triton X-100 (TM), 0.02% sodium azide) was added and is 35 bases in length and has a Tm of 76° C. Sequence 4, incubated at 72° C. for 5 minutes. The RNA:probe hybrids, from 23S rRNA, is 33 bases in length and has a Tm of 73° bound to the magnetic particles, were collected and the C. supernatant removed. One ml wash solution (0.12M sodium To demonstrate the reactivity and specificity of probe 1 phosphate buffer pH 6.8, 14% diisobutyl sulfosuccinate, 5% for members of the genus Mycobacterium, it was tested as 10 Triton X-100, 0.02% sodium azide) was added, the particles a probe in hybridization reactions under the following collected and the supernatant removed. This step was conditions. “I-labeled oligonucleotide probe was mixed repeated two times. The radioactivity bound to the magnetic with rRNA released from cells of 30 species of mycobacteria particles was determined in a gamma counter. The results are by the Sonic disruption techniques described in Murphy et al shown in Table 22 and indicate that the probes hybridize to ., U.S. patent application Ser. No. 841,860. 3x10 cells were 15 organisms in the genus Mycobacterium and that a combi suspended in 0.1 ml 5% SDS and sonicated for 15 minutes nation of probes will detect all members of the genus. Table at 50°-60° C. One ml of hybridization buffer (45% diisobu 23 shows that the probes do not react with other closely tyl sulfosuccinate, 40 mM sodium phosphate pH 6.8, 1 mM related bacteria.

TABLE 22 HYBRIDIZATION OF THE MYCOBACTERIUM PROBES 1-4TO MYCOBACTERAL SPECIES % Probe a Probe a Probe a Probe Organism ATCC # 1 Bound 2 Bound 3 Bound 4 Bound Mycobacterium africanum 25420 41.5 14.7 7.9 26.7 M. asiaticum 25274 31.8 20.2 7.9 0.1 M. avium 25291 11.7 34.7 10.1 1.6 M. bovis 19210 19.4 28.4 44.6 20.9 M. bovis (BCG) 35734 30.0 35.5 17.8 56 M. chelonae 14472 8.6 0.7 6.3 0.2 M. flavescens 14474 29.8 7.7 2.3 0.9 M. fortuitum 6841 34.7 2.2 4.8 0.2 M. gastri 5754 27.6 65. 9.6 22.3 M. gordonae 14470 50.7 55.2 3. 0.4 M. haemophilum 2.9548 40.7 60.7 0.4 12.4 M. intracellulare 13950 38.8 48.3 0.9 5.4 M. kansasii 12478 53.4 27.3 24.5 27.8 M. malnoense 29571 3. 38.4 0.8 15 M. marinun 827 4.7 4. 4.8 0.1 M. nonchromogenicum 1930 35.0 42.9 0.5 16.4 M. phlei 11758 23.7 0.6 8 0.6 M. scrofulaceum 1998 35.1 66.9 0.9 26.4 M. shinoidei 27962 34.6 1.4 1.3 4.8 M. siniae 25275 45.9 44.0 5.3 0.1 M. smegmatis el4468 31.3 4.0 5.6 0. M. szulgai 23069 19.4 22.3 15 3.0 M. terrae 15755 25.6 21.7 0.4 12.3 M. thermoresistibile 19527 20.3 34.5 3. 17.6 M. triviale 23292 37.3 4.6 4.3 0. M. tuberculosis (avirulent) 25177 38.5 26.3 1.3 23.0 M. tuberculosis (virulent) 27294 13.8 12.4 38.4 22.3 M. ulcerans 19423 33.9 28.7 0.4 8.9 M. vaccae 15483 8.8 36.2 4.8 3.2 M. Xenopi 19971 38.4 2. 3.8 0.2

TABLE 23 HYBRIDIZATION OF THE MYCOBACTERIUMPROBES 1-4. TO PHYLOGENETICALLY CLOSELY RELATED ORGANISMS

%, Probe % Probe % Probe 2. Probe Organism ATCC it 1 Bound 2 Bound 3 Bound 4 Bound Actinomadura 1942.5 0.2 0.3 0.2 0.1 madurae Actinoplanes 0049 0.4 0.5 0.3 0.2 italicus Arthrobacter 14358 0.2 0.4 0.3 0.1 oxidans 5,547,842 29 30 TABLE 23-continued HYBRIDIZATION OF THE MYCOBACTERIUM PROBES -4TO PHYLOGENETICALLY CLOSELY RELATED ORGANISMS % Probe % Probe % Probe Probe Organism ATCC it 1 Bound 2 Bound 3 Bound 4 Bound Brevibacterium c9172 0.3 0.3 0.3 0.1 linens Corynebacterium 373 0.4 0.3 0.3 0.1 erosis w Dermatophilus 14367 0.4 0.6 0.3 0.2 congolensis Microbacterium 818O 0.2 0.3 0.2 0.1 lacticum Nocardia 19247 0.3 0.3 0.4 0.1 asteroides Nocardia 19296 0.4 0.3 0.6 0.1 brasiliensis Nocardia 14629 0.4 0.4 1.0 0.3 otitidis caviarum Nocardioposis 23218 0.3 0.2 0.3 0.1 dassonvillei oerskovia 33225 0.2 0.2 0.3 0. turbata oerskovia 27402 0.2 0.3 0.3 0.1 xanthineolytica Rhodococcus 33611 0.4 0.2 0.3 0.2 aichiensis Rhodococcus 25938 0.3 0.4 0.3 0.2 aurantiacus Rhodococcus 25592 0.4 0.3 0.3 0.1 bronchialis Rhodococcus 33609 0.6 0.4 0.3 0.3 chubuensis Rhodococcus equi 6939 0.4 0.4 0.4 0.5 Rhodococcus 3360 0.5 0.5 0.3 0. obuensis Rhodococcus sputi 29627 0.4 0.5 0.4 0.3

EXAMPLE 9 capricolum (Hori, H. et al. 1981, Nucl. Acids Res. 9, Mycoplasmas are small, aerobic bacteria lacking cell 5407-5410) and Spiroplasma sp. (Walker, R.T. et al. 1982 walls. Mycoplasma pneumonias is estimated to cause 8-15 Nucl. Acids Res. 10, 6363-6367). The alignments were million infections per year. The infections may be asymp 40 performed as described above and outlined at page 6. 5S tomatic or range in severity from mild to severe bronchitis rRNA can be isolated and sequenced as outlined in Rogers and pneumonia. The organism is believed to cause about et al., or a primer can be made which is complementary to 10% of pneumonias in the general pppulation and 10-50% a conserved region in the 5S rRNA and sequencing per of the pneumonias of members of groups in prolonged, close formed as outlined in Examples 1-4. The conserved region contact such as college students and military personnel. 45 of 5S rRNA is documented in Fox, G. E. and Woese, C. R., Diagnosis until now has required isolation of the organ 1975, Nature 256: 505-507. The following sequence was ism in culture or demonstration of an increase in antibody determined to be specific for Mycoplasma pneumoniae: titer. Culturing of the organism involves inoculation of GCTTGGTGCTTTCCTATTCTCACTGAAA respiratory tract specimens onto agar or bipbasic media CAGCTACATTCGGC containing bacterial growth inhibitors. Examination for 50 The sequence is complementary to a unique segment growth at 3-4 and 7-10 days is used to establish the found in the 5S rRNA of Mycoplasma pneumoniae in the presence or absence of any mycoplasma. Mycoplasma pneu region corresponding to bases 65-108 of E. coli 5S rRNA, monias must then be identified by hemadsorption (the ability and was selected by comparison to 5S rRNA sequences from of M. pneumoniae to adhere sheep or guinea pig erythro Mycoplasma gallisepticum, Spiroplasma mirum and Urea cytes), hemolysis (the ability of M. pneumoniae to produce 55 plasma urealyticum. The oligonucleotide probe was charac beta hemolysis of sheep or guinea pig erythrocytes in blood terized as described above. The size of the probe was 42 agar), growth inhibition by specific antibodies, or immun bases. The probe has a Tm of 71.5° C. ofluorescence with specific antibodies. The present inven To demonstrate the reactivity of this sequence for Myco tion has significant advantages over each of these prior art plasma pneumoniae, the probe was tested in hybridization methods both because of the simplicity of the test and 60 reactions under the following conditions. 'P-end-labelled because of the greatly reduced time necessary to achieve a oligonucleotide probe was mixed with 1 microgram (7x10 diagnosis. 13 moles) of purified rRNA from Mycoplasma pneumoniae A probe specific for the 5S rRNA of M. pneumoniae was and reacted in 0.12M PBS (equimolar amounts of NaHPO, obtained by a comparison of known rRNA sequences. The and NaHPO), 1 mM EDTA and 0.2% SDS (sodium particular sequences aligned were from M. pneumoniae, M. 65 dodecyl sulfate) at 65° C. for 60 minutes in a final volume gallisepticum and Ureaplasma urealyticum (Rogers, M.J. et of 50 microliters. In separate tubes the probe was mixed with al. 1985, Proc, Natl. Acad. Sci. USA, 82 (1160-1164), M. the hybridization buffer with and without target Mycoplasma 5,547,842 31 32 pneumoniae rRNA present. Following separation on coli 16S rRNA. Probe sequence #1 was obtained using a hydroxyapatite as outlined previously the hybrids were quantitated by scintillation counting. These results are primer with the sequence 5'-GGCCGTTACCCCACCTAC shown in Table 24. TAGCTAAT3'. Probe sequence #2 was obtained with a primer with the sequence 5'-GTATTACCGCGGCT. TABLE 24 GCTGGC-3'. Probe sequence #3 was obtained with a primer HYBROIZATION OF THE M. PNEUMONIAE 5S rRNA with the sequence 5'-CCGCTTGTGCGGGCCCCCGT. DNA PROBE TO HOMOLOGOUSTARGET rRNA*f CAATTC-3'. Probe sequence #4 was obtained using a plus rRNA minus rRNA primer with the sequence 5'-CGATTACTAGCGATTCC-3'. 10 Sequencing reactions were performed as outlined in previ M. pneumoniae 5S probe 85-95% 0.5% ous examples. The M. pneumoniae sequences were com *% Hybridization = cpm bound to hydroxyapatite pared with sequences from Mycoplasma genitalium, Myco total cpm added to reaction plasma capricolum, Mycoplasma gallisepticum and 15 Spiroplasma mirum. This data shows that the probe has a high extent of reaction to its homologous target and very little non-specific The following probe sequences were characterized by binding to the hydroxyapatite. criteria described in example one of the parent application Specificity of the M. pneumoniae, 5S probe was tested by and were shown to be specific for Mycoplasma pneumonias: mixing the 'Plabelled probe with rRNA released from cells from other Mycoplasma species. All hybridization assays 20 2. AATAACGAACCCTTGCAGGTC were carried out as described in Example 1. Table 25 CTTTCAACTTTGAT indicates that the probe is specific for Mycoplasma pneu 3. CAGTCAAACTCTAGCCATTACCT moniae and does not react with any other Mycoplasma Species. GCTAAAGTCATT 25 4. TACCGAGGGGATCGCCCCGACAGCTAGTAT TABLE 25 5. CTTTACAGATTTGCTCACTTTTA HYBRIDIZATION OF M. PNEUMONIAE PROBETO CAAGCTGGCGAC. OTHERMYCOPLASMA SPECIES Probe #2 is 35 bases in length and has a Tm of 67° C. Probe Acholeplasma laidlawi 14089 3.3 30 #3 is 35 bases in length and has a Tm of 66° C. Probe #4 is M. buccale 23636 1.7 30 bases in length and has a Tm of 69° C. Probe #5 is 35 M. capricoium 23205 24 M. columbinsale 33549 1.4 bases long with a Tm of 66° C. M. faucium 25293 1.4 When the four probes were mixed and used in hybridiza M. fermentans 15474 1.0 tion assays at 60° C. in the same manner as previous M. gallisepticum 1960 18 35 M. gallopavonis 33551 1.6 examples, they were found to be specific for M. pneumoniae. M. genitalium 3353c 1.7 The probes do not cross react with other respiratory patho M. hominis 14027 13 M. orale 23714 1.8 gens or with any organism representing the bacterial phy M. pneumoniae 1553 18.0 logenetic tree (Table 28). M. primatum 15497 1.6 M. salivarium 23064 0.6 40 Spiroplasma mirum 2.9335 2.3 TABLE 27 HYBRIDIZATION OF MYCOPLASMA PNEUMONIAE As shown in Table 26, the probe did not react with any other PROBES 2-5 TO MYCOPLASMASPECIES closely related or phylogenetically diverse species of bac Organism ATCC is % Probe Bound teria. 45 Acholeplasma axanthum 27378 0.34 TABLE 26 Acholeplasma laidlawi 14089 0.30 Mycoplasma arcginini 23838 0.20 HYBRIDIZATION OF M. PNEUMONIAE PROBETO Mycoplasma arthritidis 19611 0.49 A PHYLOGENETIC CROSS SECTION OF BACTERIA Mycoplasma bovigenitalium 19852 0.8 50 Mycoplasma bovis 25523 0.43 Organism ATCC is % Probe Bound Mycoplasma buccale 23636 0.37 Mycoplasma californicum 33451 0.79 Corynebacterium cerosis 373 1.4 Mycoplasma capricoium 23205 0.38 Haemophilus influenzae 19418 1.4 Mycoplasma columbinasale 33549 0.54 Klebsiella pneumoniae 23357 1.3 Mycoplasma columborale 29258 0.50 Legionella pneumophia 33152 18 55 Mycoplasma faucium 25293 0.45 Mycobacterium tuberculosis (avir) 2577 1.6 Mycoplasma fermentans 5474 0.27 Mycoplasma pneumoniae 15531 52 Mycoplasma gallisepticum 19610 0.25 Neisseria meningitidis 13077 0.6 Mycoplasma gallopavonis 33551 0.47 Propionibacterium acnes 6919 2.0 Mycoplasma genitalium 33530 2.5 Pseudomonas aeruginosa 25330 1.6 Mycoplasma hominis 14027 0.52 Staphylococcus aureus 12598 2.0 60 Mycoplasma hyorhinis 17981 0.46 Streptococcus pneumoniae c6306 1.9 Mycoplasma orale 23714 0.56 Mycoplasma pneumoniae 15531 340 Mycoplasma primatum 15497 0.71 Four additional probe sequences (numbered 2-5 below) Mycoplasma pulmonis 1962 0.68 specific for Mycoplasma pneumoniae were obtained by Mycoplasma salivarium 23064. 0.46 utilizing four unique primers complementary to conserved 65 Spiroplasma citri 2946 0.60 regions on 16S rRNA. The regions correspond, respectively, Spiroplasma mirum 2.9335 0.52 to bases 190-230; 450-490; 820–860; and 1255-1290 of E. 5,547,842 33 34 5'GCT CGT TGC GGG ACT TAA CCC ACC AT-3'. TABLE 28 Sequencing with these primers was performed as described HYBREDIZATION OF MYCOPLASMA PNEUMONIAE for previous examples. PROBES 2-5 WITH OTHER BACTERIA The following three sequences were characterized by the Organism ATCC % Probe Bound criteria described in Example 1 and were shown to be specific for the genus Legionella. The phylogenetically Actinomyces israeli 10049 1.0 Bacteroides fragilis 23745 1.4 nearest neighbors Escherichia coli, Pseudomonas aerugi Bifidobacterium breve 15700 1.0 nosa, Vibrio parahaemolyticus and Acinetobacter calcoace Bordetella bronchiseptica 10580 0.9 10 ticus were used as comparisons with sequences from Clostridium innocuum 14501 1.0 Legionella species. Clostridium pasteurianum 6013 0.9 Clostridium perfringens 13124 1.1 1. TACCCTCTCCCATACTCGAGTCAACCAG Clostridium ranosum 25582 1.0 TATTATCTGACC Corynebacterium xerosis 373 0.8 Erysipelothrix rhusiopathiae 19414 1.1 2. GGATTTCACGTGTCCCGGCCTACTTGT Escherichia coli 11775 1.0 15 TCGGGTGCGTAGTTC Haemophilus influenzae 19418 0.9 Klebsiella pneumoniae 1553 1.0 3. CATCTCTGCAAAATTCACTGTATGT Lactobacillus acidophilus 4356 1.4 CAAGGGTAGGTAAGG. Legionella pneumophila 33154 0.8 Sequence 1, from 16S rRNA, is 40 bases in length and has Listeria monocytogenes 15313 1.2 aTm of 72° C. Sequence 2, from 23S rRNA, is 42 bases in Moraxella osloensis 19976 1.1 20 Mycobacterium tuberculosis 25177 1.0 length and has a Tm of 73° C. Sequence 3, from 16S rRNA, Neisseria meningitidis 13077 1.0 is 40 bases in length and has a Tm of 68°C. These sequences Pasteurella multocida 6529 1.6 are capable of hybridizing to RNA of the genus Legionella Peptococcus magnus 14955 0.9 in the regions corresponding respectively to, 630-675 of E. Propionibacterium acnes 6919 1. Pseudomonas aeraginosa 25330 1.0 coli 16S rRNA; 350-395 of E. coli 23s rRNA; and 975-1020 Staphylococcus aureus 12660 1.0 of E. coli 16s rRNA. When mixed together the probes had Streptococcus faecalis 19433 1.5 a combined average Tm of 73° C. Analysis on polyacryla Streptococcus mitis 981 1.0 mide gels showed that each probe was the correct length and Streptococcus pneumoniae 6306 1.0 sequence analysis demonstrated that each was the correct Streptococcus pyogenes 19615 1.1 sequence of bases. 30 When the three probes were mixed and used in a hybrid ization assay, they were found to be specific for the genus EXAMPLE 10 Legionella (Tables 29 and 30) and did not cross react with other respiratory pathogens or with any selected organism The genus Legionella contains 22 species which are all from the phylogenetic tree (Tables 31 and 32). Use of more potentially pathogenic for humans. These organisms cause 35 than one probe, i.e., a mixture of probes, can result in Legionnaires' disease, an acute pneumonia, or Pontiac fever, increased assay sensitivity and/or in an increase in the an acute, non-pneumonic, febrile illness that is not fatal. number of non-viral organisms to be detected. Legionella species have also been shown to be respon sible for nosocomial pneumonia occuring predominantly TABLE 29 among immunocompromised patients. 40 HYBRIDIZATION OF LEGIONELLA Legionellosis, which includes Legionnaires' disease and PROBESTO HOMOLOGOUSTARGET rRNA Pontiac fever, is diagnosed on the basis of clinical symp toms, either direct or indirect fluorescence antibody tests, plus rRNA minus rRNA and by culture using a buffered charcoal yeast extract Legionella probe 80% 1.0% (BCYE) agar containing selective antimicrobial agents. 45 There is no single definitive genus test known in the prior art. (See Bergey's Manual of Systematic Bacteriology at page 283, (ed. 1984)). The fluorescent antibody tests are not TABLE 30 able to identify all species of Legionella, but only those few HYBRIDIZATION OF LEGIONELLA for which antibodies exist. The culture method is not defini 50 PROBESTO LEGIONELLASPECIES tively diagnostic for Legionella species. Organism ATCC it % Probes Bound The oligonucleotide sequences described below, when L. anisa 35292 42.0 used as probes in a nucleic acid hybridization assay, accu L. bozemani 33217 58.0 rately identify all species of Legionella. This assay is more 55 L. cherri 35252 69.0 sensitive than culture or antibody tests and shortens signifi L. dumafi 33279 57.0 cantly the time of identification and, thus, diagnosis. The L. erythra CDC 9PWO44C 26.0 L. feelei 35303 59.0 assay, therefore, represents a significant improvement over L. hackeliae 35250 47.0 prior diagnostic methods. L. jamestowniensis 35298 20.0 Three probe sequences specific for the genus Legionella L. jordanis 33623 50.6 L. longbeachae 33484 48.0 were obtained by utilizing three unique primers complemen L. maceacherni 35300 25.0 tary to conserved regions on both 16S and 23S rRNA. L. micaadei 33704 38.0 Sequence 1 was obtained by using a 16S primer with the L. oakridgensis 33761 440 sequence 5'-TCT ACG CAT TTC ACC GCT ACA C-3'. L. parisiensis 9060 69.0 L. pneumophila 1 6736 75.0 Probe sequence 2 was obtained with a 23S primer of 65 L. pneumophila 2 640 sequence 5'-CAGTCAGGA GTATTT AGCCTT3'. Probe L. pneumophila 3 73.0 sequence 3 was obtained with a 16S primer of sequence 5,547,842 35 36 The following three sequences were characterized by the TABLE 30-continued criteria previously described and were shown to be specific HYBRIDIZATION OF LEGIONELA for the genus Legionella. The phylogenetically nearest PROBESTO LEGIONELLASPECIES neighbors Escherichia coli, Pseudomonas aeruginosa, Vibrio parahaemolyticus and Actinetobacter calcoaceticus Organism ATCC i. % Probes Bound were used for comparisons with sequences from Legionella L. pneumophila 4 73.0 species. L. pneumophila 5 78.0 4. GCG GTA CGGTTCTCTATA AGT TAT GGCTAG L. pneumophila 6 75.0 C L. pneumophia 7 73.0 O L. pneumophila 8 63.0 5. GTA CCG AGG GTA CCTTTG TGCT L. pneumophila II 75.0 L. rubrittcens 35304 12.0 6. CAC TCTTGG TAC GAT GTC CGA C L. Sainthelensi 35248 610 Probe 4, complementary to 23S rRNA in the region L. Sainticrucis 353O 24.0 corresponding to bases 1585-1620 of E. coli 23S rRNA, is L. spiritensis CDCMSH9 55.0 L. Steigerwaiti 7430 56.0 15 31 bases long and has a Tm of 67° C. Probe 5, complemen L. wadsworthi 33877 370 tary to 23S rRNA in the region corresponding to bases 2280-2330 of E. coli 23S rRNA, is 22 bases long and has a *The numbers 1-8 and 11 are serotypes of L. pneumophila. Tm of 66° C. Probe 6, complementary to 23S rRNA in the same region as Probe 5, is 22 bases long and has a Tm of 63 TABLE 31 20 C. When the three probes were mixed with probe 3 above HYBRIDIZATION OF LEGONELLA PROBESTO and used in a hybridization assay as described for probes RESPIRATORY PATHOGENS 1-3, they were found to be specific for the genus Legionella Organisms ATCC is % Probe Bound (Table 33) and did not cross react with other respiratory 25 pathogens or with any selected organism from the phyloge Corynebacterium rerosis 373 2. Haemophilus influenzae 19418 2.3 netic tree (Tables 34 and 35). using more than one probe, i.e., Klebsiella pneumoniae 23357 2.0 a mixture of probes, can improve assay sensitivity and/or Mycoplasma pneumoniae 15531 2.3 increase the number of non-viral organisms detected. Neisseria meningitidis 13090 2.2 Pseudomonas aeruginosa 25330 12 TABLE 33 Propionibacterium acnes 6919 1.6 30 Streptococcus pneumoniae 6306 0.8 HYBRDIZATION OF LEGIONELLA PROBESTO Staphylococcus aureus 25923 6 LEGONELLASPECIES Organism ATCC i % Probes Bound TABLE 32 35 L. anisa 35292 29.6 L. bozemani 33217 35.5 HYBRIDIZATION OF LEGIONELLAPROBESTO L. Cherri 35252 29.2 A PHYLOGENETIC CROSS SECTION OF L. dunofi 33279 26.0 BACTERIAL SPECIES L. erythra 35303 32.0 L. feeli CDC9P1WO44C 32.0 Organisms ATCC is % Probe Bound 40 L. hackeliae 352.50 39.0 L. jamestowniensis 35298 31.2 Acinetobacter calcoaceticus 33604 14 L. jordanis 33623 25.7 Branhanella catarrahalis 25238 2.0 L. longbeachae 33484 27.6 Bacius subtilis 6051 19 L. maceaherni 35300 39.3 Bacteroides fragilis 23745 2.2 L. micaladei 33204 31.0 Campylobacter jejuni 33560 1.2 45 L. Oakridgensis 3376 24.4 Chronobacterium violaceum 29094 1.3 L. parisiensi 35299 31.2 Clostridium perfringens 13124 1.9 L. pneumophila I* 3353 40.0 Deinoccoccus radiodurans 35073 1.8 L. pneumophila 2 33154 38.5 Dercia gunmosa 15994 2.0 L. pneumophila 3 3355 44.6 Enterobacter aerogenes 1304.8 1.4 L. pneumophila 4 33.56 48.6 Escherichia coli 11775 1.2 L. pneumophila 5 33216 32.0 Mycoplasma hominis 14027 1.1 50 L. pneumophila 6 33215 43.0 Proteus mirabilis 2.9906 14 L. pneumophila 7 33823 29.5 Pseudomonas cepacia 1762 1.1 L. pneumophila 8 35096 37.6 Rahnella aquatis 3307 1.7 L. pneumophila II 43130 445 Rhodospirillum rubrum 170 2.0 L. rubriucers 35304 30.1 Streptococcus mitis 9811 2.0 L. sainthelensis 35248 27.0 Vibrio parahaemolyticus 17802 20 55 L. sainticrucis 35301 22.0 Yersinia enterocalitica 9610 2 L. spiritensis CDCFEMSH9 40.5 L. steigerwaiti 35302 31.7 Three additional probe sequences (numbered 4-6) spe L. wadsworthi 33877 30.0 cific for the genus Legionella were obtained by utilizing two *The numbers 1-8 and 11 are serotypes of L. pneumophila. primers complementary to conserved regions on 23S rRNA. 60 Sequence 4 was made from a 23S primer with the sequence TABLE 34 5'-CCTTCTCCC GAA GTT ACG G-3'. Probe sequences 5 HYBRIDIZATION OF LEGIONELLA PROBESTO and 6 were made from a 23S primer of sequence 5'-AAG RESPIRATORY PATHOGENS CCG GTT ATCCCC GGG GTA ACTTTT-3". Sequencing 65 Organisms ATCC % Probe Bound with these primers was performed as described for previous Corynebacterium xerosis 373 0.3 examples. 5,547,842 37 38 say of clinical specimens. The method of choice, however, TABLE 34-continued remains culture of the organism in cycloheximide treated HYBRIDIZATION OF LEGIONELLA PROBESTO McCoy cells. Cell culture is followed by morphological or RESPIRATORY PATHOGENS fluorescent antibody staining for confirmation of the organ 5 Organisms ATCC it % Probe Bound ism's identity. Haemophilum influenzae 19418 0.12 The inventive oligonucleotide sequences described Klebsiella pneumoniae 23357 0.13 below, when used as probes in nucleic acid hybridization Neisseria meningitidis 13090 0.14 Pseudomonas aeruginosa 25330 0.13 assay, accurately identify Chlamydia trachomatis isolates. Propionibacterium acnes 6919 0.11 10 This assay test is equal in sensitivity to culture or antibody Streptococcus pneumoniae 6306 0.08 tests and, in the case of culture, significantly shortens the Staphylococcus aureus 25923 0.15 time to identification, and thus, diagnosis. The use of probes to identify and distinguish between members of the species is novel and inventive. Indeed, TABLE 35 15 Kingsbury, D. T., and E. Weiss, 1968 J. Bacteriol. HYBRIDIZATION OF LEGIONELLA PROBESTO A PHYLOGENETIC CROSS SECTION OF 96:1421–23 (1968); Moulder, J. W., ASM News, Vol.50, BACTERIAL SPECIES No.8, (1984) report a 10% DNA homology between C. trachomatis and C. psittaci. Moreover, these reports show Organisms ATCC if 7. Probe Bound that different C. trachomatis strains differ in DNA homology, 20 Acinetobacter calcoaceticus 33604 0.12 Weisberg, W. G. et al, J. Bacteriol. 167:570–574 (1986) Branhamella catarrahalis 25238 0.13 published the 16S rRNA sequences of C. psittaci and noted Bacillus subtilis 6051 0.09 that C. trachomatis and C. psittaci share a greater than 95% Bacteroides fragilis 23745 0.12 Campylobacter jejuni 33560 0.06 rRNA homology. From these reports, it may be inferred that Chromobacterium violaceum 29094 0.33 25 it would be difficult to invent (1) probes capable of hybrid Clostridium perfringens 13124 0.07 izing to all strains of C. trachomatis; and (2) probes capable Deinoccoccus radiodurans 35073 0.11 Dercia gunmosa 15994 0.5 of distinguishing between C. trachomatis and C. psittaci. Enterobacter aerogenes 13048 0.26 The following probes accomplish both objectives. Escherichia coli 11775 0.09 Ten probe sequences specific for Chlamydia trachomatis Mycoplasma hominis 14027 0.09 30 Proteus mirabilis 2.9906 0.09 were made using seven unique primers complementary to Pseudomonas cepacia 17762 0.20 conserved regions of both 16S and 23S rRNA. Probe Rahnella aquatilis 33071 0.15 sequence 1 was obtained from a 16S primer of sequence Rhodospirillum rubrum 11170 0.13 Streptococcus mitis 9811 0.07 5'-TCT ACG CAT TTC ACC GCT ACA C-3', Probe Vibrio parahaemolyticus 17802 0.11 sequence 2 was obtained with a 16S primer of sequence Yersinia enterocolitica 9610 0.19 35 5'-CCG CTT GTG CGG GCC CCC GTC AAT TC-3'. Sequences 3 and 4 were obtained using a 16S primer with t the sequence 5'-GGCCGT TAC CCC ACC TACTAG CTA EXAMPLE 11 AT-3'. Probe sequences 5 and 6 were obtained with a 23S Chlamydia are gram-negative, non-motile, obligate intra- 40 primer of sequence 5'-CTTTCC CTCACG GTA-3'. Probe cellular bacteria. The species C. trachomatis is associated sequences 7 and 8 were obtained with a 23S primer of with endemic trachoma (the most common preventable form sequence 5'-CCTTCT CCC GAA GTT ACG G-3. Probe of blindness), inclusion conjunctivitis and lymphogranu sequence 9 was obtained with a 23S primer of sequence loma venereum (LGV). It is a major cause of nongonococcal 5'-TCG GAACTTACC CGA CAAGGAATTTC-3'. Probe urethritis in men and may cause cervicitis and acute salp- 45 sequence 10 was obtained with a primer of sequence 5'-CTA ingitis in women. Eye disease or chlamydial pneumonia may CTTTCCTGC GTCA-3'. develop in newborns passing through the infected birth The following ten sequences were characterized using the canal. criteria described in Example 1 and were shown to be There are several methods known in the art for identifi specific for the rRNA of Chlamydia trachomatis. The phy cation of C. trachomatis in the urogenital tract, for example, logenetically nearest neighbor Chlamydia psittaci was used by direct immunofluorescent staining or enzyme immunoas for comparison with Chlamydia trachomatis sequence.

1. CCG ACT CGG GGT TGA GCC CAT CTT TGA CAA 2. TTA CGT CCG ACA CGG ATG GGGTTG. AGA CCA TC 3. CCG CCA CTA AAC AAT CGT CGA AAC AAT TGC TCC GTT CGA 4. CGT TAC TCG GAT GCC CAA ATA TCG CCA CAT TCG 5. CAT CCA TCT TTC CAG ATG TGT TCA ACT AGG AGT CCT GAT CC 6. GAG GTC GGT CTT TCT CTC CTT TCG TCT ACG 7. CCG TTC TCA TCG CTC TAC GGA CTC TTC CAATCG 8. CGA AGA TTC CCC TTG ATC GCG ACC TGA TCT 5,547,842 39 40 -continued 9, CCG GGG CTC CTA TCG TTC CAT AGT CAC CCT AAA AG 10. TAC CGC GTG TCT TAT CGA CAC ACC CGC G 5 Sequence 1, from 16S rRNA, is 30 bases in length and has aTm of 66° C. Sequence 2, from 16S rRNA, is 32 bases in TABLE 37 length and has a Tm of 67° C. Sequence 3, from 16S rRNA, is 39 bases in length and has a Tm of 70° C. Sequence 4, HYBRIDIZATION OF CHLANYDIA TRACHOMATIS from 16S rRNA, is 33 bases in length and has a Tm of 69 10 PROBES3-9 WITH CHLAMYDIA rRNA C. Sequence 5, from 23S rRNA, is 41 bases in length and has Ratio Counts aTm of 71° C. Sequence 6, from 23S rRNA, is 30 bases in Organisms Serovar ATCC Bound length and has a Tm of 72° C. Sequence 7, from 23S rRNA, C. trachomatis A 689 is 33 bases in length and has a Tm of 72° C. Sequence 8, 15 C. trachomatis B 560 from 23S rRNA, is 30 bases in length and has a Tm of 71° C. trachomatis Ba 1066 C. trachomatis C WR548 962 C. Sequence 9, from 23S rRNA is 35 bases in length and has C. trachomatis D 1192. a Tm of 74° C. Sequence 10 is 28 bases in length and has C. trachomatis E VR348 1022 a Tm of 72° C. C. trachomatis F 391 20 C. trachomatis G WR878 874 The reactivity and specificity of the probes was tested C. trachomatis H 954 hybridization assays. 'P-end-labeled oligonucleotide C. trachonatis I WR880 943 C. trachomatis J 482 probes 1 and 2 were mixed with purified RNA or RNA C. trachomatis K VR887 999 released from at least 10' organisms in 0.55 ml of 41% C. trachomatis Ll 638 diiSobutyl sulfosuccinate, 3% sodium dodecyl sulfate, C. trachomatis L2 501 25 C. trachonatis L3 WR903 821 0.03M sodium phosphate pH 6.8, 1 mM EDTA, 1 mM C. psittaci VR125 1.6 EGTA at 60° C. (probe 1) or 64° C. (probe 2) for 1 hour. C. psittaci VR629 0.9 Hybrids were bound to hydroxyapatite as described in C. psittaci VR656 1.3 previous examples and the amount of radioactivity bound C. psittaci VR813 1.2 was determined by scintillation counting. Table 36 shows 30 *Ratio = counts bound when RNA present that probes 1 and 2 hybridize well to all serotypes of C. counts bound when no RNA present trachomatis tested. Probe 1 does not react with any strain of C. psittaci tested and probe 2 does not react with two of the strains. Probe 2 does react with the ovine polyarthritis strain TABLE 38 of C. psittaci, an organism which is not known to infect 35 HYBRDIZATION OF CHLAMYDIA TRACHOMATIS humans. Table 37 demonstrates the reactivity and specificity PROBES3-9 TO ORGANISMS FOUND IN THE of probes 3-9 when 'I-labeled and used as a mix. In this UROGENITAL TRACT, case, the hybrids were bound to cationic magnetic particles Ratio Counts as described in Arnold et al., U.S. patent application Ser. No. 40 Organism ATCC Bound 020,866 filed Mar. 2, 1987. These probes hybridize well to Achromobacter xylosoxidans 27061 19 all strains of C. trachomatis tested and not to any strains of Acinetobacter lwoffii 15309 1.2 C. psittaci. Probes 3-9 were further tested against a panel of Branhamella catarrhalis 25238 1.2 organisms commonly found in the ufogenital tract (Table 38) Candida albicans 18804 2.4 and a phylogenetic cross section of organisms (Table 39). In Flavobacterium meningosepticum 13253 1.1 45 Gardnerella vaginalis 14018 1.3 all cases, the probes were shown to be specific. Probe 10 is Lactobacillus acidophilus 4356 0.8 25% non-homologous to C. psittaci and also should be Listeria monocytogenes 15313 0.7 specific for C. trachomatis. Mycobacterium smegmatis 14468 1.1 Moracella osloensis 19976 1.3 Neisseria gonorrhoeae 19424 2.3 TABLE 36 50 Pasteurella multocida 6529 1.0 Peptostreptococcus anaerobius 27331 1.2 HYBRIDIZATION OF CHLAMYDIA TRACHOMATIS Streptococcus agalactiae 13813 4.0 PROBES AND 2 TO CHLAMYDIA RNA Streptococcus faecalis 19433 2.6 % Probe Bound *Ratio = Counts bound when RNA present Probe Probe 55 counts bound when no RNA present Organism ATCCi 2 Chlamydia trachomatis serotype C VR578 22 39 TABLE 39 Chlamydia trachonatis serotype E WR348B 27 48 Chlamydia trachomatis serotype G WR878. 20 44 HYBRIDIZATION OF CHLAMYDIA TRACHOMATIS Chlamydia trachomatis serotype I WR880 20 42 60 PROBES3-9 TO PHYLOGENETICALLY DIVERSE Chlamydia trachomatis serotype K VR887 28 45 ORGANISMS. Chlamydia psittaci guinea pig WR813 1.2 1.4 conjunctivitis strain Ratio Counts Chlamydia psittaci ovine VR656 10 3.0 Organism ATCCh Bound abortion strain Chlamydia psittaci ovine poly- VR619 11 35.3 65 Bacillus subtilis 6051 2.2 arthritis strain Bacteroides fragiles 23745 1.6 Campylobacter jejuni 33560 1.4 5,547,842 41 42 haemolyticus and Wolinella succinogenes were used for TABLE 39-continued comparison with the campylobacter sequences. HYBRIDIZATION OF CHLAMYDIA TRACHOMATIS 1. CGCTCC GAAAAGTGT CAT CCT CC PROBES3-9 TO PHYLOGENETICALLY DIVERSE 2. dCTTAG GTA CCG TCA GAATTC TTC CC ORGANISMS. 3. GCC TTC GCA ATG GGTATT CTTGGT G Ratio Counts 4. GGTTCTTAG GAT ATC AAG CCC AGG Organism ATCC# Bound Sequence 1, from 16S rRNA, is 23 bases in length and has Chromabacterium violaceum 29094 1.4 aTm of 65° C. Sequence 2, from 16S rRNA, is 26 bases in Deinococcus radiodurans 35073 1.8 10 length and has a Tm of 64° C. Sequence 3, from 16S rRNA, Derxia gummosa 15994 1.3 is 25 bases in length and has a Tm of 66° C. Sequence 4, Enterobacter aerogenes 13048 1.9 from 16S rRNA, is 24 bases in length and has a Tm 61° C. Escherichia coli 11775 19 Sequence 1 is capable of hybridizing in the region corre mycoplasma hominis 14027 1.3 Pseudomonas cepacia 17762 2.2 sponding to bases 405-428 of E. coli 16S rRNA; Sequence Proteus mirabilis 2.9906 2.2 2 is capable of hybridizing in the region corresponding to Rahnella aquatilis 33071 19 15 bases 440-475 of E. coli 16s rRNA; Sequence 3 is capable Rhodospirillum rubrum 11170 1.9 of hybridizing in the region corresponding to bases 705-735 Vibrio parahaemolyticus 17802 2.0 of E. coli 16s rRNA; Sequence 4 is capable of hybridizing Yersinia enterocolitica 9610 2.5 in the region corresponding to bases 980-1010 of E. coli 16s rRNA *Ratio = Counts bound when RNA present 20 The reactivity and specificity of the probes for campylo counts bound when no RNA present bacter was tested in hybridization assays. 'P-end-labeled oligonucleotide probes were mixed with purified RNA or RNA released from cells in 0.1% sodium dodecyl sulfate. EXAMPLE 12 0.5 ml of hybridization solution (41% diisobutyl sulfosuc cinate, 30 mM sodium phosphate, pH 6.8, 0.7% sodium Campylobacters are motile, microaerophilic, gram nega dodecyl sulfate, 1 mM EDTA, 1 mM EGTA) was added and tive curved rods. The genus is quite diverse and distinct from the mixture incubated at 60° C. for 1 to 1.5 hour. Following other genera. Although the genus is well defined, some incubation, 2 to 2.5 ml of separation solution (2% hydroxya revision is occurring at the species level (Romaniuk, P.J. et patite, 0.12M sodium phosphate, pH 6.8, 0.02% sodium al., J. Bacteriol. 169:2137-2141 (1987). Three Campylo dodecyl sulfate) was added and the mixture incubated at 60 bacter species, Campylobacter jejuni, C. coli and C. laridis, 30 C. for five minutes. The sample was centrifuged and the cause enteritis in humans. The disease includes diarrhea, supernatant removed. 2.5 ml of wash solution (0.12M fever, nausea, abdominal pain and in some cases, vomiting. sodium phosphate, pH 6.8, 0.02% sodium dodecyl sulfate) These organisms cause an estimated 2 million infections per was added and the sample mixed, centrifuged and the year in the United States (estimate based on the number of sUpernatant removed. The radioactivity bound to the Salmonella and Shigella induced cases of diarrheal disease). 35 hydroxyapatite was determined by scintillation counting. Other members of the genus cause septicemias in humans Table 40 indicates that the probes hybridize well to the and abortion and infertility in sheep and cattle. Campylobacter species of interest, C. jejuni, C. coli, and C. Diagnosis of Campylobacter enteriris is currently depen laridis. Probe 1 detects all of the Campylobacter species dent upon growth and isolation of the organism in culture, tested, probes 2 and 4 detect only the enteric campylo followed by a number of biochemical tests. Optimum 40 bacters, and probe 3 detects all of the Campylobacter species growth of campylobacters requires special conditions such except C. sputorum, an organism isolated from cattle. Thus as low oxygen tension and high temperature (42° C). No all of the probes are useful for identifying campylobacter in single set of conditions is recommended for isolation of all stool samples. The choice of which probe to use for other Campylobacter species. applications would depend upon the level of specificity 45 required (i.e., enteric campylobacters, or all Campylobacter The oligonucleotide sequences listed below, when used in species). a hybridization assay, hybridize to the 16S rRNA of the Campylobacter species of interest. The present invention has TABLE 40 significant advantages over the prior art methods of detec tion of Campylobacter because one probe can detect all HYBRDIZATION OF CAMPYLOBACTER PROBES 1-4 Campylobacters of interest; the other two probes detect the 50 TO CAMPYLOBACTERSPECIES. enteric Campylobacters and one can detect human isolates % Probe Bound (%) of Campylobacter. In addition, the probes have advantages over the prior art in terms of ease of the assay and greatly Organism ATCC 2 3 4. reduced time to identification and therefore, diagnosis. 55 Campylobacter coli 33559 64 70 52 49 The four probes which hybridize to the 16S rRNA of C. fetus 27374 68 0. 66 0.5 Campylobacter species of interest were constructed using subsp. fetus C. fetus 19438 66 0.7 54 1.2 three unique primers complementary to 16S rRNA. subsp. venerealis Sequences 1 and 2 were made using a 16S primer with the C. jejuni 33560 63 76 51 56 sequences 5'-GTATTA CCG CGG CTG CTG GCA C-3'. 60 C. laridis 35221 74 73 64 52 Sequence 3 was made using a 16S primer with the sequence C. sputorum 33562 71 3.0 2.5 O 5'-CCG CTT GTG CGG GCC CCC GTC AAT TC-3'. subsp. bubulus Sequence 4 was made with a 16S primer with the sequence (*) % Probe Bound= cpm bound to hybroxyapatite-cpm bound when no RNA 5'-GCT COST TGC GGG ACT TAA CCC AAC AT3'. present/total cpm used in the assay The following sequences were characterized and shown to 65 Table 41 shows that the probes do not hybridize to closely hybridize to Campylobacter jejuni, C. coli and C. laridis. related organisms or organisms found in the gastrointestinal The phylogenetically nearest neighbors Vibrio para tract. 5,547,842 43 44 homology. (Kiepper-Baez, 1981, 1982, Schliefer 1984.) The TABLE 41 current invention also reduces the number of tests which HYBRIDIZATION OF CAMPYLOBACTER PROBES 1-4 must be run on a sample and greatly reduces the time to TO CLOSELY RELATED ORGANISMS AND ORGAN identification and thus, diagnosis. This represents a signifi ISMS FOUND IN THE GASTRONTESTINAL TRACT, 5 cant improvement over prior art methods. The probe sequence was identified using a primer comple % Probe Bound (%) mentary to 16S rRNA with the sequence 5'-CCGCTT GTG Organism ATCC 1 2 3 4 CGG GCC CCC GTCAATTC-3'. The following sequence was characterized and shown to be specific for three entero Bacteroides fragiles 25285 O 0.2 0.7 0 10 Escherichia coli 11775 113 0.5 0.5 O cocci, S. faecium, S. faecalis and S. avium. The phyloge Salmonella typhimurium 14028 0 O 0.3 0 netically nearest neighbors S. agalactiae, S, bovis, Spneu Shigella boydii 29929 O 0.2 0.5 0 moniae and S. pyogenes used for comparison with the Shigella dysenteriae 13313 0 0.7 0.2 0 Shigella flexneri 299.03 0 O 0.5 0 sequences of interest. Shigella sonnei 29930 0 O 0.1 O 1. TGC AGC ACT GAA GGG CGG AAA CCC TCC Vibrio parahaemolyticus 17802 0 1.9 0.1 0 15 AAC ACTTA Wollinella succinogenes 29.543 0.4 2. 2.2 0 The sequence is 35 bases in length and has a Tm of 72 Yersinia pseudotuberculosis 29833 0.6 0.2 1.7 0.3 C. It is capable of hybridizing in the region corresponding to (*) % probe bound= cpm bound to hydroxyapatite-cpm bound when no RNA bases 825-860 of E. coli 16S rRNA. To demonstrate the present/total cpm used in the assay reactivity and specificity of the probe, it was used in a The probes specific for the enteric Campylobacters probes 2 20 hybridization assay with purified RNA or RNA released and 4, were further tested and shown not to react with from cells. A suspension containing at least 10" cells in 2% rRNAs of other organisms found in the gastrointestinal tract. sodium dodecyl sulfate was vortexed in the presence of glass beads. 0.1 ml of suspension was mixed with 0.1 ml of TABLE 42 hybridization buffer (0.96M sodium phosphate, pH 6.8, 25 0.002M EDTA, 0.002M EGTA) and incubated at 65° C. for HYBRIDIZATION OF CAMPYLOBACTER PROBES 2 AND 4 TO ORGANISMS FOUND IN THE 2 hours. After incubation, 5 ml of 2% hydoxyapatite, 0.12M GASTROINTESTINAL TRACT sodium phosphate pH 6.8, 0.02% sodium dodecyl sulfate was added and the mixture was incubated at 65° C. for 10 % Probe Bound (%) minutes. The sample was centrifuged and the Supernatant 30 removed. Five ml of wash solution (0.12M phosphate buffer, Organism ATCCi Probe 2 Probe 4 pH 6.8, 0.02% sodium dodecyl sulfate) was added and the Citrobacter diversus 271.56 O O samples were vortexed, centrifuged, and the supernatant Clostridium perfringens 13124 O O removed. The amount of radioactivity bound to the Enterobacter cloacae 13047 0 O Klebsiella pneumonias 23357 O 0.5 35 hydroxyapatite was determined by scintillation counting. Proteus mirabilis 25933 O 0. Table 43 shows that the probe reacts well with S. faecium, Serratia marcescens 13880 O 0. S. faecalis, and S. avium, and does not react with other Staphylococcus aureus c12600 O 0. closely related organisms. Staphylococcus epidermidis 14990 O 0.3 Streptococcus bovis 33317 O O TABLE 43 40 (*) % probe bound= cpm bound to hydroxyapatite-cpm bound when no RNA HYBRDIZATION OF THE ENTEROCOCCUS PROBE present/total cpm used in the assay TO CLOSELY RELATED ORGANISMS. Organism ATCCh % Probe Bound

EXAMPLE 13 45 Staphylococcus aureus 12600 1.4 Streptococcus agalactiae 1383 1.5 Streptococci are gram positive, oxidase negative coccoid Streptococcus avium 14025 22.7 Streptococcus bovis 33317 14 bacteria. The genus has been divided into 18 groups, A-R, on Streptococcus faecalis 19433 45.3 the basis of group-specific carbohydrates. Group D strepto Streptococcus faecium 19434 43.0 cocci are further subdivided into the enteroccocci (S. 50 Streptococcus mitis 9811 1.5 faecium, S. faecalis, S. avium and S. gallinarum and the Streptococcus pneumonias 6306 15 nonenterococci S. bovis and S. equinus. Sfaecium, S. faeca Streptococcus pyogenes 19615 1.3 lis and S. avium are considered the medically important enteroccocci. Some species of streptococcus are human EXAMPLE 14 pathogens; others are normal flora in the mouth and intestine 55 but are capable of causing disease when introduced to other Pseudomonads are gram-negative, nonsporeforming, sites. Two examples are S. faecium and S. faecalis which are nonfermentative bacilli. Pseudomonads are common inhab normally found in the intestine but may spread to cause itants of soil and water and rarely infect healthy individuals. bacteremia, wound infections, and as many as 10% of the When the organisms encounter already compromised urinary tract infections in the United States. 60 patients, they can cause a variety of clinical syndromes Current methods of detection of enterococci require cul including wound infections, postsurgical infections, septi ture of the specimen for 18-72 hours followed by a battery cemia, infant diarrhea and respiratory and urinary tract of biochemical tests. The oligonucleotide sequence shown infections. Members of the genus pseudomonas are particu below, when used in a hybridization assay, accurately larly important to identify in a clinical sample because of the detects Streptococcus faecalis, S. avium, and S. faecium. The 65 resistance of the organisms to antibiotics. Nucleic acid inventive probe does not cross react with other Streptococci homology studies have divided the genus into five homology or Staphylococci which are very closely related in DNA classes known as RNA groups I-V. Eighty-three percent of 5,547,842 45 46 all clinical isolates of Pseudomonas are from RNA group I and Pseudomonas aeruginosa is by far the most common TABLE 45-continued species isolated. HYBRIDIZATION OF PSEUDOMONAS GROUPI Current methods of detection of pseudomonas require PROBETO RNAs OF CLOSELY RELATED ORGANISMS 5 culture of a patient sample for 24-72 hours, followed by a % Probe battery of biochemical tests. The oligonucleotide sequence Organism ATCC Bound below, when used in a hybridization assay, detects the Legionella pneumophila 33155 0.6 clinically important group I pseudomonas. The present Moraxella phenylpyruvica 23333 0.3 invention reduces the number of tests which must be run on 10 Morganella morgani 25830 O a sample, and reduces the time to detection. This represents Vibrio parahaemolyticus 17802 0.6 a significant improvement over prior art methods. *% Probe Bound = counts bound when RNA present - counts bound when no The sequence was obtained with a primer complementary RNA present/total counts used in the assay to a conserved region on 23S rRNA with the sequence 5'-CTTTCC CTC ACG GTA-3'. The following sequence 15 EXAMPLE 1.5 was shown to detect group I pseudomonads: 1. CAGACAAAGTTT CTC GTG CTC CGT CCT ACT Examples 15-18 disclose probes for the Enterobacteri CGATT aceae, all of which are highly related at the DNA level. Even The probe is 35 bases in length and has a Tm of 70° C. fewer differences exist at the rRNA level. For example, It is capable of hybridizing to the RNA of group I 20 Proteus vulgaris 16S rRNA is 93% homologous to E. coli. Pseudomonas in the region corresponding to bases 365-405 These factors illustrate the difficulties associated with mak of E. coli 23s rRNA. To demonstrate the reactivity and ing rRNA probes specific for this group of organisms. specificity of the probe, it was used in a hybridization assay. Nevertheless, we have invented probes for Enterobacter 'P-end-labeled oligonucleotide was mixed with RNA cloacas, Proteus mirabilis, Salmonella and E. coli. released from at least 10' organisms by standard methods in 25 Members of the genus Enterobacter are motile, gram 0.48M sodium phosphate pH 6.8, 1% sodium dodecyl sul negative, non-sporeforming bacilli which belong in the fate, 1 mM EDTA, 1 mM EGTA and incubated at 65° C. for family Enterobacteriaceae. The genus is a large and hetero two hours. After incubation, the RNA:DNA hybrids were geneous group. Eight species have been defined but only 5 bound to hydroxyapatite as described for previous examples are clinically significant. Enterobacter cloacae and E. aero and the radio-activity bound was determined by scintillation 30 genes are the most common isolates and are associated with counting. Table 44 demonstrates that the probe reacted well genitourinary, pulmonary, blood, central nervous system and with all 8 species of group I. pseudomonads that were tested. soft tissue infections in humans. The probe did not react with RNA from group II or group V The current method for identifying Enterobacter cloacae organisms. A low reaction was seen with Pseudomonas from patient samples involves culture of the specimen on acidovorans, a group III organism which represents <1% of agar plates for 18-24 hours, followed by a battery of all isolates of nonfermentative bacilli from clinical samples. biochemical tests. The oligonucleotide sequence described Table 45 demonstrates that the probe does not react with below, when used as a probe in a nucleic acid hybridization other closely related organisms which were tested. assay, accurately identifies Enterobacter cloacas. The present invention reduces the number of tests which must be TABLE 44 40 run on a sample, the time to identification and therefore, HYBRIDIZATION OF PSEUDOMONAS GROUPI diagnosis, and thus represents a significant improvement PROBE TOPSEUDOMONAS RNAs over prior art methods. % Probe The probe specific for Enterobacter cloacas was obtained Organism Group ATCCi Bound 45 with a primer complementary to a conserved region of 23S rRNA with the sequence 5'-CAGTCAGGA GTATTTAGC Pseudomonas alcaligenes I 4909 24 CTT.'3. Pseudomonas aeruginosa I 1045 83 Pseudomonas denitrificans I 3867 83 The following sequence was. characterized and shown to Pseudomonas fluorescens I 13525 82 be specific for E. cloacae. The phylogenetically nearest Pseudomonas mendocina I 2541 79 50 neighbors Escherichia coli, Klebsiella pneumoniae, Proteus Pseudomonas pseudoalcaligenes I 17440 78 Pseudomonas putida 12633 8O vulgaris, Salmonella enteritidis, and Citrobacter freundi Pseudomonas stutzeri I 17588 84 were used as comparisons with the sequence of E. cloacae. Pseudomonas cepacia 25416 O 1. GTG TGT TTT CGT GTA CGG GAC TTT CAC CC Pseudomonas picketti II 2751 O Pseudomonas acidovorans III 5668 The probe is 29 bases in lengthand has a Tm of 68° C. It Pseudomonas maltophilia V 13637 0.2 55 is capable of hybridizing to RNA of E. cloacae in the region corresponding to bases 305-340 of E. coli 238 rRNA. To *% Probe Bound = counts bound when RNA present counts bound when no demonstrate the reactivity and specificity of the probe for E. RNA present/total counts used in the assay cloacae, it was used in a hybridization assay, 'P-end labeled oligonucleotide probe was mixed with RNA released TABLE 45 60 from at least 10' organisms in 1% sodium dodecyl sulfate, HYBRIDIZATION OF PSEUDOMONAS GROUPI 0.48M sodium phosphate, pH 6.8 (0.2 ml final volume) and PROBE TO RNAS OF CLOSELY RELATED ORGANISMS incubated at 60° C. for 2 hours. Following incubation, 5 ml of 2% hydroxyapatite, 0.12M sodium phpsphate pH 6.8, Probe 0.02% sodium dodecyl sulfate was added and the mixture Organism ATCC Bound 65 incubated at 60° C. for 10 minutes. The sample was centri Acinetobacter calcoaceticus 23055 1.6 fuged and the supernatant removed. Five ml of wash solu tion (0.12M sodium phosphate, pH 6.8, 0.02% sodium 5,547,842 47 48 dodecyl sulfate) was added, the sample vortexed, centri The probe specific for Proteus mirabilis was obtained fuged and the supernatant removed. The amount of radio with a primer complementary to a conserved region of 23S activity bound to the hydroxyapatite was determined by rRNA with the sequence 5'-CAGTCAGGA GTATTT AGC scintillation counting. The results are shown in Table 46 and CTT3'. demonstrates that the probe reacts well with E. cloacae and The following sequence was characterized and shown to does not react with the RNA of closely related organisms. be specific for P. mirabills. The phylogenetically nearest TABLE 46 neighbors Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris and Salmonella enteritidis were used as compari HYBRIDIZATION OF ENTEROBACTER CLOACAE 10 sons with the sequence of Proteus mirabills. PROBE TO CLOSELY RELATED ORGANISMS 1. CCGTTCTCC TGA CAC TGCTATTGATTA AGA % Probe CTC Organisms Name ATCC# Bound This probe is capable of hybridizing to the RNA of P. Citrobacter greundi 8090 18 mirabilis in the region corresponding to base 270–305 of E. Enterobacter aerogenes 13048 1.4 15 coli 23s rRNA. The probe is 33 bases in length and has a Tm Enterobacter cloacae 13047 27. of 66° C. To demonstrate the reactivity and specificity of the Escherichia coli 11775 1.0 Klebsiella pneumoniae 13883 .7 probe for P. mirabilis, it was used in a hybridization assay. Proteus mirabilis 2.9906 0.9 'P-end-labeled oligonucleotide probe was mixed with RNA Proteus vulgaris 1335 0.6 released from at least 10' organisms in 1% sodium dodeoyl Providencia stuarti 29914 1.1 20 sulfate, 0.48M sodium phosphate, pH 6.8, 1 mM EDTA, 1 mMEGTA (0.2 mi final volume) and incubated at 64° C. for Table 47 shows that the probe does not react with the RNA 2 hours. Following incubation, 5 ml of 2% hydroxyapatite, of organisms found in urine. 0.12M sodium phosphate pH 6.8, 0.02% sodium dodecyl sulfate was added and the mixture incubated at 64° C. for 10 TABLE 47 25 minutes. The sample was centrifuged and the supernatant removed. Five ml of wash solution (0.12M sodium phos HYBRIDIZATION OF ENTEROBACTER CLOACAE phate, pH 6.8, 0.02% sodium dodecyl sulfate) Was added, PROBE TO ORGANISMS FOUND IN URINE. the sample vortexed, centrifuged and the supernatant was % Probe removed. The amount of radioactivity bound to the Organisms Name ATCC Bound 30 hydroxyapatite was determined by scintillation counting. The results are shown in Table 48 and demonstrate that the Candida albicans 18804 0.8 Candida krusei 34135 0.8 probe reacts well with P. mirabilis and does not react with Candida parapsilosis 22019 0.9 27 other closely related bacteria. Table 49 shows that the Candida tropicalis 750 1.1 Pseudomonas aeruginosa 101.45 10 probe does not react with 24 other phylogenetically diverse Serratia marcenscens 13880 1.6 35 bacteria and two yeasts tested in the same manner as the Staphylcoccus aureus 2600 1.7 organisms in Table 48. Staphylococcus epidermidis 14990 1.4 Streptococcus agalactieae 1383 2.5 TABLE 48 Streptococcus faecium 19434 15 Torulopsis glabrata 2001 0.9 HYBRIDIZATION OF PROTEUS MIRABILIS PROBE 40 TO CLOSELY RELATED ORGANISMS % Probe Organism Name ATCC Bound EXAMPLE 16 Citrobacter diversus 271.56 1.1 Members of the genus Proteus are motile, gram negative, 45 Citrobacter freundi 8090 1.1 non-sporeforming bacilli which belong in the family Entero Citrobacter freundi 6750 1.0 Enterobacter aerogenes 13048 1.0 bacteriaceae. Four species of Proteus have been described Enterobacter agglomerans 27155 1.0 and three of them, Proteus mirabilis, P. vulgaris, and P. Enterobacter cloacae e13047 1.1 penneri, cause human disease. Enterobacter gergoviae 33028 1.0 Enterobacter sakazaki 29544 1.1 The most common type of proteus infection involves the 50 Escherichia coli 10798 1.2 urinary tract, but septicemia, pneumonia and wound infec Escherichia coli 11775 1.2 tions also occur. Proteus mirabilis is the species most often Escherichia coli 2.94.17 1.2 isolated and may account for up to 10% of all acute, Klebsiella oxytoca 13182 1.0 Klebsiella ozaenae 11296 1.1 uncomplicated urinary tract infections. Species, rather than Klebsiella planticola 33531 0.9 genus level identification of the causative organism is desir 55 Klebsiella pneumoniae 13883 1.3 able because of differential antibiotic susceptibility among Klebsiella pneumoniae 23357 1.1 the species. Klebsiella rhinoscleromates 13884 1.2 Klebsiella terrigena 33257 1.1 The current method for identifying Proteus mirabills from Klebsiella trevisani 33558 10 patient samples involves culture of the specimen on agar Kluyvera ascorbata 33433 0.9 plates for 18-24 hours, followed by a battery of biochemical 60 Proteus mirabilis 25933 69.0 Proteus penneri 33519 2.5 tests. The oligonucleotide sequence described below, when Proteus vulgaris 13315 1.7 used as a probe in a nucleic acid hybridization assay, Providencia alcalifaciens 9886 1.1 accurately identifies Proteus mirabilis. The present inven Providencia rettgeri 29944 1.3 tion reduces the number of tests which must be run on a Providencia stuartii 29914 1. sample, the time to identification and therefore, diagnosis 65 Salmonella arizonae 29933 1.1 and treatment. This represents a significant improvement Salmonella enteritidis 13076 0.8 over prior art methods. 5,547,842 49 50 Sequence 2 was obtained using a 23S primer with the TABLE 49 sequence 5' CAG TCA GGA GTATTT AGC CTT3'. The HYBRDIZATION OF PROTEUS MIRABILIS PROBETO following sequences were characterized and shown to be PHYLOGENETICALLY DIVERSE ORGANISMS specific for the genus Salmonella: % Probe 1. CTC CTTTGA GTT CCC GAC CTAATCGCT GGC Organism Name ATCCit Bound 2. CTCATC GAG CTCACA CCA CAT GCGCTTTTG Acinetobacter calcoaceticus 33604 0.8 TGT A Bacilius subtilis 6051 1.2 Sequence 1, from 16S rRNA, is 30 bases in length and has Bacteroides fragilis 23745 0.9 10 aTm of 73° C. Sequence 2, from 23s rRNA, is 34 bases long Branhamella catarrhalis 25238 0.7 Campylobacter jejuni 33560 1.0 and has a Tm of 71° C. These probes are capable of Candida krusei 34135 0.8 hybridizing in the regions corresponding to bases Chromobacterium violaceum 29094 1.1 1125-1155 of E. coli 16S rRNA and 335-375 of E. coli 23s Clostridium perfringens 13124 0.9 Deinococcus radiodurans 35073 0.8 rRNA, respectively. To demonstrate the reactivity and speci Derxia gummosa 15994 0.8 15 ficity of probe 1 for members of the genus Salmonella, Hafnia alvei 13337 0.9 'P-end-labeled oligonucleotide was tested as a probe in a Morganella morgani 25830 0.9 hybridization reaction. Purified RNA, or RNA released from Pseudomonas aeruginosa 101.45 1.0 Pseudomonas cepacia 17762 0.9 at least 10' organisms by standard.methods, was mixed with Rahnella aquatilis 33071 0.9 1 ml hybridization buffer (final concentration 43% diisobu Rhodospirillum rubrum 11170 0.8 20 tyl sulfosuccinate, 60 mM sodium phosphate pH 6.8, 1 mM Serratia marcescens 3880 0.9 EDTA, 1 mM EGTA) and incubated at 72° C. for 2-12 Serratia odorifera 33077 0.9 Staphylococcus aureus e12600 0.8 hours. Following incubation, 5 ml of separation solution Staphylococcus epideridis 14990 0.8 (2% hydroxyapatite, 0.12M sodium phosphate, pH 6.8, Streptococcus mitis 9811 0.8 0.02% sodium dodecyl sulfate) was added and the sample Streptococcus pneumoniae e6306 0.9 25 were mixed, incubated at 72° C. for 5 minutes, centrifuged Torulopsis glabrata 2001 0.9 Vibrio parahaemolyticus 17802 0.8 and the supernatants removed. Four ml of wash solution Xanthomonas maltophilia 13637 1.1 (0.12M sodium phosphate pH 6.8, 0.02% sodium dodecyl Yersinia enterocolitica 9610 0.8 sulfate) was added and the samples were vortexed, centri fuged, and the supernatants removed. The amount of radio 30 activity bound to the hyproxyapatite was determined by scintillation counting. The results shown in Table 50 indicate EXAMPLE 17 that a combination of the two probes hybridized to the 5 Members of the genus Salmonella are motile, gram nega subgroups of Salmonella and to all 31 of the serotypes which tive, non-sporeforming bacilli which belong in the family were tested. Enterobacteriaceae. All salmonellae are highly related and 35 some microbiologists consider them to be one species. Five TABLE 50 subgroups have been identified using nucleic acid homology HYBRIDIZATION OF SALMONELLA PROBES 1 AND 2 studies and over 1400 different serotypes have been TO MEMBERS OF THE GENUS SALMONELLA described. All serotypes have been implicated in human % Probe Bound enteric disease ranging from selflimited gastroenteritis with 40 mild symptoms, to severe gastroenteritis with bacteremia, to Subgroup Organism ATCCi Probe 1 Probe 2 typhoid fever, a potentially life-threatening illness. S. chol I Salmonella 10708 24 40 erasuis, S. paratyphi A and S. typhi are the serotypes most choleraesuis often associated with severe disease and bacteremia. Diag I Salmonella enteritidis 13076 15 67 nosis of Salmonella-induced enteriris is dependent upon 45 I Salmonella paratyphi 9150 1.4 70 A detection of the organism in stool samples. Because infec I Salmonella sp. serotype 92.70 40 26 tion occurs primarily by ingestion of contaminated milk, datum food and water, methods for identifying Salmonella in these Salmonella sp. serotype 12007 54 35 products before release to consumers is critical. cubana 50 I Salmonella sp. serotype 9268 12 40 Current methods for detection of members of the genus give Salmonella involve culture of the specimen for 1-3 days on I Salmonella sp. serotype 8326 53 33 selective media followed by a battery of biochemical tests. heidelberg I Salmonella sp. serotype 1646 36 46 Often an enrichment step is needed to isolate Salmonella illinois from clinical samples or food products. The oligonucleotide 55 I Salmonella sp. serotype 8387 35 32 sequences shown below, when used in a hydridization assay, montevideo I Salmonella sp. serotype 29628 52 34 accurately identify members of the genus Salmonella. The newington present inventive probes are specific for all members of the I Salmonella sp. serotype 6962 34 36 genus and do not react with the other closely related Entero newport bacteriaceae genera. These inventive probes reduce the 60 I Salmonella sp. serotype 15787 34 39 number of tests which must be run on a sample and greatly putten I Salmonella sp. serotype 9712 28 30 reduce the time to identification. This represents a significant saintpault improvement over prior art methods. I Salmonella sp. serotype 8400 38 43 The probes specific for the genus Salmonella were senftenberg I Salmonella sp. serotype 2004 29 29 obtained with two primers complementary to 16S and 23S 65 simsbury rRNA. Sequence 1 was obtained using a 16S primer with the I Salmonella sp. serotype 15791 34 30 sequence 5' TTA CTA GCG ATT CCG ACT TCA 3'. 5,547,842 51 52 TABLE 50-continued TABLE 52-continued HYBRIDIZATION OF SALMONELLA PROBES 1 AND 2 HYBRIDIZATION OF SALMONELLA PROBES 1 AND 2 TO MEMBERS OF THE GENUS SALMONELLA TO RNA OF A PHYLOGENETIC CROSS SECTION OF ORGANISMS % Probe Bound % Probe Bound Subgroup Organism ATCCit Probe 1 Probe 2 Organism ATCCi Probe 1 and Probe 2 sloterdijk Salmonella sp. serotype 8391 32 41 10 Campylobacter jejuni 33560 O 0.2 thompson Candida krusei 34135 0.4 0.3 I Salmonella sp. serotype 15611 35 2.6 Chromobacterium violaceum 29094 1.7 0 veilore Clostridium perfringens 13124 0.3 O I Salmonella typhi 19430 7.0 21 Deinococcus radiodurans 35073 1.6 0.1 I Salmonella 14028 69 69 Derxia gummosa 15994 1.2 0. typhimurium Hafnia alvei 13337 1.8 O II Salmonella alamae 6959 3.0 46 15 Morganelli morgani 25830 O .1 I Salmonella sp. 15793 6.6 30 Pseudomonas aeruginosa O145 0.5 0.7 serotype maarssen Pseudomonas cepacia 17762 O 0 III Salmonella arizonae 33952 2.9 38 Pseudomonas maltophilia 13637 1.9 O III Salmonella arizonae 12324 5.5 42 Rahnella aquatilis 33071 1.2 0.3 III Salmonella arizonae 29933 2.3 62 Rhodospirilium rubrum 11170 0.9 O III Salmonella arizonae 29934 63 12 20 Serratia marcescens 13880 O O III Salmonella arizonae 12323 4.0 39 Serratia odorifera 33077 2.6 0.2 III Salmonella arizonae 12325 51 19 Staphylococcus aureus c12600 0.2 O IV Salmonella sp. serotype 15783 5.8 8.0 Staphylococcus epidermidis 14990 O O harmelen Streptococcus mitis 9811 1.2 0.7 IV Salmonella sp. serotype 29932 7.5 40 Streptococcus pneumoniae e6306 O O ochsenzoll 25 Torulopsis glabrata 2001 O O W Salmonella sp. serotype cdc1319 60 1.8 Vibrio parahaemolyticus 17802 O 0.2 bongor Yersinia enterocolitica 960 O O *% Probe Bound = Counts bound to hydoxyapatite - counts bound when no The specificity of the probes for members of the genus RNA present/total counts used in assay Salmonella was demonstrated with hybridization reactions 30 containing RNA from organisms closely related to Salmo nella. The results are shown in Table 51. EXAMPLE 18 TABLE 51 Escherichia coli is a gram negative, nonsporeforming 35 bacillus which belongs in the family Enterobacteriaceae. HYBRIDIZATION OF SALMONELLA PROBES AND 2 Five species of Escherichia have been described: E. coli, TO RNA OF CLOSELY RELATED ORGANISMS which accounts for >99% of the clinical isolates, E. herma nii, E. blattae, E. vulneris and E. fergusonii, E. coli is a % Probe Bound leading cause of urinary tract infections, bactermia and Organism ATCC Probe 1. Probe 2 neonatal meningitidis, and can cause a type of gastroenteritis 40 known as traveller's diarrhea. Citrobacter freundi 6750 2.2 O Edwardsiella tarda 15947 O O The current method for identifying E. coli from patient Enterobacter agglomerans 27155 0.6 O samples involves culture of the specimen on agar plates for Enterobacter cloacae 13047 O O 18–72 hours, followed by a battery of biochemical tests on Enterobacter sakazki 29544 O O Escherichia coli 10798 O O 45 isolated colonies. The oligonucleotide sequence described Escherichia coli 2.94.17 0 O below, when used as a probe in a nucleic acid hybridization Klebsiella pneumoniae 23357 0.7 O assay, accurately detects E. coli even in the presence of other Kluybera ascorbata 33433 O 0.5 organisms. The present invention reduces the number of Proteus mirabilis 25933 0.2 O Shigella flexneri 29903 O O tests which must be run on a sample and reduces the time to 50 identification and therefore diagnosis and treatment. This *% Probe Bound = counts bound to hydroxyapatite - counts bound when no represents a significant improvement over prior art methods. RNA present/total counts used in assay The probe specific for E. coli was derived from the Table 52 shows that Salmonella probes 1 and 2 do not published E. coli sequence (Brosius, et al, Proc. Natl. Acad. hybridize to phylogenetically diverse organisms. Sci. U.S.A. 75:4801-4805 (1978)), using Proteus vulgaris 55 (Carbon, et al., Nuc. Acids Res, 9:2325-2333 (1981)), Kleb TABLE 52 siella pneumoniae, Salmonella enteritidis, Enterobacterger HYBRIDIZATION OF SALMONELLA PROBES 1 AND 2 goviae and Citrobacter freundii for comparison. The probe TO RNA OF A PHYLOGENETIC CROSSSECTION sequence is shown below. OF ORGANISMS 1. GCA CATTCT CAT CTCTGAAAACTT CCGTGG 60 It hybridizes to RNA of E. coli in the region of 995-1030 % Probe Bound of 16s rRNA. The probe is 30 bases in length and has a T. Organism ATCC# Probe and Probe 2 of 66° C. To demonstrate the reactivity and specificity of the probe for E. ocli, it was used in a hybridization assay. Acinetobacter calcoaceticus 33604 1. 0.1 Bacillus subtilis 6051 O 0.5 'P-end-labeled oligonucleotide probe was mixed with two Bacteroides fragilis 23745 0.1 0. 65 unlabeled oligonucleotides of sequence 5'-TGG ATG TCA Branhamella catarrhalis 25238 0.9 0 AGA CCA GGTAAG GTT CTT CGC GTT GCA TCG-3' and 5'-CTG ACG ACA GCC ATG CAG CAC CTG TCT 5,547.842 53 54 CAC GGTTCC CGA AGG CA-3' and with purified RNA, are harmful and cause disease. The presence of any bacteria or RNA released from cells with detergent and heat, in 1% in some locations is undesirable or indicative of disease sodium dodecyl sulfate (SDS), 0.48M sodium phosphate pH (e.g., culture media, pharmaceutical products, body fluids 6.8, 1 mM EDTA, 1 mM EGTA (0.2 ml final volume) and such as blood, urine or cerebrospinal fluid, and tissue incubated at 60° C. for 2 hours. Following incubation, 5 ml biopsies). Low levels of bacteria are considered acceptable of 2% hydroxyapatite, 0.12M sodium phosphate pH 6.8, in other products such as drinking water and food products. 0.02% sodium dodecyl sulfate was added and the mixture Accordingly, there is a need, for a means for detecting and incubated at 60° C. for 10 minutes. The sample was centri quantitating bacteria in a sample. fuged and the supernatant removed. Five ml of wash solu The current method of detection and quantitation of total tion (0.12M sodium phosphate, pH 6.8, 0.02% sodium 10 bacteria in a sample requires culture on multiple types of dodecyl sulfate) was added, the sample vortexed, centri media under different conditions of temperature and atmo fuged and the supernatant was removed. The amount of sphere. To date, no single test exists to detect or quantitate radioactivity bound to the hydroxyapatite was determined by all bacteria. The oligonucleotide sequences shown below, scintillation counting. when used in a hybridization assay, detect a broad phylo An example of a use for this probe would be to detect E. 15 genetic cross section of bacteria. The present invention coli in urine samples. Table 53 shows that the probe detects reduces the number of tests which need to be performed and 7 out of 8 strains of E. coli tested. The probe also reacts with also reduces the time required for the assay. Comparison of E. fergusonii, an organism which would only rarely be found the hybridization results from an unknown sample to a set of in urine. standards will allow some quantitation of the number of Table 54 shows that the probe does not react with any 20 bacteria present. This represents a significant improvement other genus tested except Shigella, another organism rarely overPrior art methods. isolated from urine. These results show that the probe will be The bacterial probes were designed following examina useful in detecting E. coli from urine samples. tion of published sequences of rRNA and sequences deter mined at Gen-Probe. The sequences used for the comparison TABLE 53 25 include Agrobacterium tumefaciens (Yang et. al., Proc. Natl. HYBRIDIZATION OF E. coi TO ESCHERICHIASPECIES Acad. Sci. U.S.A., 82:4443, (1985), Anacystis nidulans (Tomioka and Sugiura. Mol. Gen. Genet. 191:46, (1983), Organism ATCC % Probe Bound Douglas and Doolittle Nuc. Acids Res., 12:3373, (1984), Escherichia coli 10798 70 Bacillus subtilis (Green et al., Gene 37:26. (1985), Bacillus E. coli 1775 67 30 stearothermophilus (Kop et al., DNA 3:347, (1984), E. coli 23722 58 E. coli 25404 68 Bacteroides fragilis (Weisburg et al., J. Bacteriol. 164:230, E. coli 25922 55 (1985), Chlamydia psittaci (Weisburg et al., J. Bacteriol. E. coli 29417 72 167:570. (1986)), Desulfovibrio desulfuricans (Oyaizu and E. coli 33780 0.8 Woese, System. Appl. Microbiol. 6:257, (1985); Escherichia E. coli 35.50 45 35 E. fergusonii 35469 55 coli, (Brosius et al., Proc. Natl. Acad. Sci. U.S.A. 77:201, E. hermani 33650 0.7 (1980); Flavobacterium heparinum (Weisburg et al., J. E. vulneris 33821 0.8 Bacteriol. 164:230, (1985); Hellobacterium chlorum (Woese et al., Science 229:762, (1985); Mycoplasma PG50 40 (Frydenberg and Christiansen, DNA 4:127, (1985); Proteus TABLE 54 vulgaris is (Carbon et al., Nuc. Acids Res. 9:2325, (1981); Pseudomonas testosteroni (Yang et al., Proc. Natl. Acad, HYBRDIZATION OF THE E. coli PROBETO Sci. U.S.A. 82:4443, (1985); Rochalimaea guintana (Weis CLOSELY RELATED ORGANISMS burg et al., Science 230:556, (1985); Saccharomyces cerevi Organism ATCC % Probe Bound siae (Rubstov et al., Nuc. Acids Res. 8:5779, (1980); Geor 45 giev et al., Nuc. Acids Res. 9:6953, (1981); and human Citrobacter freundi 6750 0.8 Citrobacter freundi 8090 0.9 (Torczynski et al., DNA 4:283, (1985); Gonzalez et al., Proc. Citrobacter freundi 29221 0.6 Natl. Acad. Sci. U.S.A. 82:7666, (1985)). Citrobacter freundi 33128 0.6 The following sequences were shown to hybridize to a Enterobacter aerogenes 13048 1.2 Enterobacter agglomerans 27155 0.9 50 broad phylogenetic cross section of bacteria and not to yeast Enterobacter cloacae 13047 0.9 or human rRNA: Enterobacter gergoviae 33023 0.7 1. CCA CTG CTG CCT CCC GTAGGA GTCTGG GCC Enterobacter Sakazaki 29544 0.6 Klebsiella oxytoca 13182 0.7 2. CCA GAT CTCTAC GCATTT CAC CGCTACACG Klebsiella pneumoniae 13883 0.7 TGG Protus mirabilis 2.9906 0.7 55 Proteus vulgaris 13315 0.8 3. GCT CGT TGC GGG ACT TAA CCC AACAT Shibella boydii 8700 76 4. GGGGTT CTTTTC GCCTTT CCC ETCACGG Shigella dysenteriae 13313 0.8 5. GGCTGC TTC TAAGCC AAC ATC CTG Shigella flexneri 299.03 7 Shigella sonnei 29930 75 6. GGA CCGTTATAG TTA CGG CCGCC 60 7. GGTCGGAACTTACCC GACAAG GAATTTCGC TAC C EXAMPLE 19 Probe 1 is 30 bases long and has a Tm of 70° C. Probe 2 is 33 bases long and has a Tm of 69° C. Probe 3 is 26 bases The bacteria encompass a morphologically and physi long and has a Tm of 67° C. Probe 4 is 27 bases long and ologically diverse group of unicellular organisms which 65 has a Tm of 69° C. Probe 5 is 24 bases long and has a Tm occupy most natural environments. Although many bacteria of 66° C. Probe 6 is 23 bases long and has a Tm of 62° C. are harmless or beneficial to their environment or host, some Probe 7 is 34 bases long and has a Tm of 66° C. Probes 1-3 5,547,842 SS 56 hybridize to 16S rRNA in the following regions, respec tively, (corresponding to E. coli bases) 300-365; 675-715; TABLE 55-continued and 1080-1110. Probes 4-7 hybridize to 23S rRNA in the HYBRIDIZATION OF BACTERIAL PROBE 1 folKowing regions, respectively, (corresponding to E. coli TO RNA OF ORGANISMS FOUND IN URINE bases) 460-490; 1050-1080; and 1900–1960 (probes 6 and 7). The oligonucleotides interact with regions on the rRNA % Probek which are highly conserved among eubacteria. This means Organism ATCCit Bound that they can be used as bacterial probes in a hybridization Streptococcus faecium 19434 53 assay. A second use is as a tool to obtain rRNA sequence. For Torulopsis glabrata 2001 2.3 example, an oligonucleotide can be hybridized to the rRNA 10 Ureaplasma urealyticum 27618 54 of interest and extended with reverse transcriptase. The sequence of the resulting DNA can be determined and used to deduce the complementary rRNA sequence as described TABLE 56 in the Detailed Description of the Invention. HYBRDIZATION OF BACTERIAL PROBE 1 TO RNAs One application of the invention is to detect bacteria in 15 OF ACROSSSECTION OF PHYLOGENETICALLY urine (bacteriuria). To demonstrate the reactivity and speci DIVERSE ORGANISMS. ficity of the probes for bacteria found in urine, they were used in hybridization assays. 'P-end-labeled or 'I-labeled % Probe oligonucleotide probes were mixed with RNA released from Organism ATCCi Bound cells by standard methods (e.g., the sonic disruption tech 20 Acinetobacter calcoaceticus 23055 65 niques described in Murphy et al., U.S. patent application Bacilius subtilis 6051 73 Bacteroides fragiles 23745 61 Ser. No. 841,860, detergent with glass beads, or enzymatic Branhanella catarrhalis 25238 72 lysis). Probe was mixed with RNA in 0.48M sodium phos Campylobacter jejuni 33560 64 phate, pH 6.8, 1 mM EDTA, 1 mM EGTA, 1% sodium Chlamydia trachomatis VR878 14 dodecyl sulfate (0.2 ml final volume) and hybridized at 60° Chronobacterium violaceum 29094 71 Clostridium perfringens 1324. 74 C. for 2 hours. Five ml of 2% hydroxyapatite, 0.12M sodium Corynebacterium xerosis 373 38 phosphate pH 6.8, 0.02% sodium dodecyl sulfate was added Deinococcus radiodurans 35073 47 and the mixture incubated at 60° C. for 10 minutes. The Derxia gummosa 15994 65 mixture was centrifuged and the supernatant removed. Five Gardnerella vaginalis 14.018 67 Hafnia alvei 13337 60 ml of wash solution (0.12M sodium phosphate, pH 6.8, 30 Lactobacillus acidophilus 4356 56 0.02% sodium dodecyl sulfate) was added and the sample Moracelia osloensis 19976 61 was mixed, centrifuged and the supernatant removed. The Mycobacterium smegmatis 14468 47 amount of radioactivity bound to the hydroxyapatite was Mycoplasma hominis 14027 58 Neisseria gonorrhoeae 19424 58 determined by scintillation counting. Tables 55-68 demon Rahnella aquatilis 33071 74 strate the specificity of these probes and show that a com 35 Rhodospirilium rubrum 11170 73 bination of probes could be used to detect all bacteria which Vibrio parahaemolyticus 17802 75 have been tested. Human 2.5 Table 55 shows that probe 1 hybridizes to the RNA of bacteria commonly isolated frome urine and does not detect Table 57 shows that Probe 2 hybridizes to the RNA of yeast RNA. Table 56 shows that probe 1 detects phyloge 40 bacteria commonly found in urine except Ureaplasma ure netically diverse bacteria and does not hybridize to human alyicum and does not hybridize to yeast rRNA. RNA. TABLE 57 TABLE 55 HYBRDIZATION OF BACTERIAL PROBE 2 HYBRIDZATION OF BACTERIAL PROBE 1 45 TO RNA OF ORGANISMS FOUND IN URINE TO RNA OF ORGANISMS FOUND IN URINE % Probe % Probc Organism ATCC Bound Organism ATCCit Bound Candida albicans 18804 2.5 Candida albicans 18804 2.6 50 Candida krusei 34135 1.8 Candida krusei 34135 2.2 Candida parapsilosis 22019 1.6 Candida parapsilosis 22019 2.9 Candida tropicalis 750 1.4 Candida tropicalis 750 2.5 Citrobacter freundi 8090 61 Citrobacter freundi 8090 69 Enterobacter aerogenes 13048 57 Enterobacter aerogenes 13048 70 Enterobacter cloacae 1304.7 61 Enterobacter cloacae 13047 71 55 Escherichia coli 1775 67 Escherichia coli 1775 67 Klebsiella oxytoca 1382 67 Klebsiella oxytoca 13182 70 Klebsiella pneumoniae 13883 5. Klebsiella pneumoniae 13883 72 Morganella morgani 25830 69 Morganella morgani 25830 66 Proteus mirabilis 299.06 69 Proteus mirabilis 2.9906 71 Proteus vulgaris 13315 69 Proteus vulgaris 13315 67 60 Providencia stuartii 29914 66 Providencia stuarti 29914 69 Pseudomonas aeruginosa 101.45 59 Pseudomonas aeruginosa 101.45 76 Pseudomonas fluorescens 13525 58 Pseudomonas fluorescens 13525 73 Serratia marcescens 13880 64 Serratia marcescens 13880 66 Staphylococcus aureus 12600 60 Staphylococcus aureus 12600 57 Staphylococcus epidermidis 14990 60 Staphylococcus epidermidis 14990 68 Streptococcus agalactiae 13813 54 Streptococcus agalactiae 13813 68 65 Streptococcus faecalis 19433 37 Streptococcus faecalis 19433 51 Streptococcus faecium 19434 58 5,547,842 57 58 TABLE 57-continued TABLE 59-continued HYBRIDIZATION OF BACTERIAL PROBE 2 HYBRIDIZATION OF BACTERIAL PROBE 3 TO RNA OF TO RNA OF ORGANISMS FOUND IN URINE ORGANISMS FOUND IN URINE. % Probe % Probe Organism ATCC# Bound Organism ATCC Bound Torulopsis glabrata 2001 1.5 Streptococcus agalactiae 13813 34 Ureaplasma urealyticum 27618 3.2 Streptococcus faecalis 19433 20 10 Streptococcus faecium 19434 47 Torulopsis glabrata 2001 19 Table 58 shows that probe 2 detects phylogenetically diverse Ureaplasma urealyticum 27618 26 bacteria and does not hybridize to human rRNA. TABLE 58 Table 60 shows that probe 3 detects phylogenetically diverse 15 bacteria and does not hybridize to human rRNA. HYBRIDIZATION OF BACTERIAL PROBE 2 TO RNAs OF ACROSS SECTION OF PHYLOGENETICALLY TABLE 60 DIVERSE ORGANISMS. HYBRIDIZATION OF BACTERIAL PROBE 3 TO RNAs % Probek OF ACROSSSECTION OF PHYLOGENETICALLY Organism ATCC Bound 20 DIVERSE ORGANISMS. Acinetobacter calcoaceticus 23055 76 % Probe Bacillus subtilis 6051 75 Organism Name ATCC Bound Bacteroides fragiles 23745 2.0 Branhamella catarrhalis 25238 70 Acinetobacter calcoaceticus 23055 69 Campylobacter jejuni 33560 2.5 Bacillus subtilis 6051 35 Chlamydia trachomatis VR878 16 Bacteroides fragiles 23745 1.2 chromobacterium violaceum 29094 61 Branhamella catarrhalis 25238 43 Clostridium perfringens 13124 66 Campylobacter jejuni 33560 55 Corynebacterium xerosis 373 3.8 Chlamydia trachomatis VR878 42 Deinococcus radiodurans 35073 6.0 Chromobacteriula violaceum 29094 69 Derxia gummosa 15994 61 Clostridium perfringens 13124 62 Gardnerella vaginalis 14018 2.0 30 Corynebacterium xerosis 373 23 Hafnia alvei 13337 72 Deinococcus radiodurans 35073 30 Lactobacillus acidophilus 4356 50 Deria gunmosa 15994 67 Moracella osloensis 19976 64 Gardnerella vaginalis 1408 40 Mycobacterium smegmatis 14468 19 Hafnia alvei 13337 56 Mycoplasma hominis 14027 34 Lactobacillus acidophilus 4356 36 Neisseria gonorrhoeae 19424 71 35 Moracella osloensis 19976 64 Rahnella aquatilis 33071 77 Mycobacterium smegmatis 14468 77 Rhodospirillum rubrum 11170 1.5 Mycoplasma hominis 14027 1.5 Vibrio parahaemolyticus 17802 73 Neisseria gonorrhoeae 19424 26 ersinia enterocolitica 9610 76 Rahnella aquatilis 33071 66 Human 2.0 Rhodospirillum rubrum 1170 51 40 Vibrio parahaemolyticus 17802 68 Yersinia enterocolitica 9610 68 Table 59 shows that probe 3 hybridizes to the RNA of Human 0.9 bacteria commonly found in urine and does not detect yeast rRNA. Table 61 shows that probe 4 hybridizes to the RNA of TABLE 59 45 bacteria commonly found in urine and does not detect yeast rRNA. HYBRIDIZATION OF BACTERIAL PROBE 3 TO RNA OF ORGANISMS FOUND IN URINE. TABLE 61 % Probe HYBRDIZATION OF BACTERAL PROBE 4 TO RNA OF Organism ATCC Bound 50 ORGANISMS FOUND IN URINE.

Candida albicans 18804 1.4 % Probe Candida krusei 34135 1.5 Organism ATCCi Bound Candida parapsilosis 22019 2.2 Candida tropicalis 750 2.6 Candida albicans 18804 4.5 Citrobacter freundi 8090 79 55 Candida krusei 34135 2.5 Enterobacter aerogenes 13048 40 Candida parapsilosis 2209 2.7 Enterobacter cloacae 13047 44 Candida tropicalis 750 2.5 Escherichia coli 11775 67 Citrobacter freundi 8090 55 Klebsiella oxytoca 13182 38 Enterobacter aerogenes 13048 52 Klebsiella pneumoniae 13883 45 Enterobacter cloacae 13047 57 Morganella morgani 25830 57 60 Escherichia coli 1775 70 Proteus mirabilis 2.9906 40 Klebsiella oxytoca 13182 70 Proteus vulgaris 13315 51 Klebsiella pneumoniae 13883 43 Providencia stuarii 29914 54 Morganella morgani 25830 74 Paeudomonas aeruginosa 101.45 61 Proteus mirabilis 2.9906 74 Pseudomonts fluoreacens 13525 56 Proteus vulgaris 13315 73 Serratia mancescens 13880 54 Providencia stuartii 29914 73 Staphylococcus aureus 12600 37 65 Pseudomonas aeruginosa 101.45 76 Staphylococcus epidermidis 14990 20 Pseudomonas fluorescens 13525 79 5,547,842 59 60

TABLE 61-continued TABLE 63-continued HYBRDIZATION OF BACTERIAL PROBE 4TO RNA OF HYBRIDIZATION OF BACTERIAL PROBE 5TO RNA OF ORGANISMS FOUND IN URINE. ORGANISMS FOUND IN URINE. 5 % Probe % Probe Organism ATCC: Bound Organism ATCC Bound Serratia marcescens 13880 74 Providencia Stuartii 29914 44 Staphylococcus aureus 12600 73 Pseudomonas aeruginosa 101.45 47 Staphylococcus epidermidis 14990 73 10 Pseudomonas fluorescens 13525 25 Streptococcus agalactiae 13813 70 Serratia narcescens 13880 35 Streptococcus faecalis 19433 37 Staphylococcus aureus 12600 26 Streptococcus faecium 19434 63 Staphylococcus epidermidis 14990 37 Torulopsis glabrata 2001 2.2 Streptococcus agalactiae 1383 29 Ureaplasma urealyticum 27618 43 Streptococcus faecalis 19433 14 15 Streptococcus faecium 19434 33 Torulopsis glabrata 2001 2.2 Table 62 shows that probe 4 detects phylogenetically diverse Ureaplasma urealyticum 27618 73 bacteria and does not hybridize to human rRNA. Table 64 shows that probe 5 detects phylogenetically diverse TABLE 62 bacteria and does not hybridize to human RNA. HYBRIDIZATION OF BACTERIAL PROBE 4 TO RNAs OFACROSS SECTION OF PHYLOGENETICALLY TABLE 64 DIVERSE ORGANISMS HYBRDIZATION OF BACTERIAL PROBE 5TO RNAS OF % Probe ACROSS SECTION OF PHYLOGENETICALLY DIVERSE Organism Name ATCC Bound 25 ORGANISMS Acinetobacter calcoaceticus 23055 69 % Probe Bacillus subtilis 6051 55 Organism ATCC Bound Bacteroides fragilis 23745 3.0 Branhamella catarrhalis 25238 59 Acinetobacter calcoaceticus 23055 20 Campylobacter jejuni 33560 65 Bacillus subtilis 6051 53 Chlamydia trachomatis VR878 50 30 Bacteroides fragilis 23745 44 Chromobacterium violaceum 29094 61 Branhanella catarrhalis 25238 22 Clostridium perfringens 13124 57 Campylobacter jejuni 33560 35 Corynebacterium xerosis 373 9.5 Chronobacterium violaceum 29094 59 Deinococcus radiodurans 35073 63 Clostridium perfringens 3124 63 Derxia gummosa 15994 65 Corynebacterium xerosis 373 1.7, Gardnerella vaginalis 14018 57 35 Deinococcus radiodurans 35073 5.7 Hafnia alvei 13337 67 Deraia gummosa 15994 14 Lactobacillus acidophilus 4356 68 Gardnerella vaginalis 14.018 1.6 Moraxella osloensis 19976 68 Hafnia alvei 13337 44 Mycobacterium smegmatis 14468 28 Lactobacillus acidophilus 4356 1.5 Mycoplasma hominis 14027 74 Moraxella oaioeksis 19976 7.2 Neisseria gonorrhoeae 19424 76 40 Mycobacterium smegmatis 14468 39 Rahnella aquatilis 33071 68 Mycoplasma hominis 14027 21 Rhodospirillum rubrum 11170 59 Neisseria gonorrhoeae 19424 40 Vibrio parahaemolyticus 17802 75 Rahnella aquatilis 33071 55 Yersinia enterocolitica 9610 74 Rhodospirillum rubrum 11170 17 Human 2.8 Vibrio parahaemolyticus 17802 66 45 Yersinia enterocolitica 9610 64 Table 63 shows that probe 5 hybridizes to the RNA of Human 1.6 bacteria commonly found in urine and does not detect yeast Table 65 shows that probe 6 hybridizes to the RNA of rRNA. bacteria commonly found in urine and does not detect yeast TABLE 63 50 rRNA. HYBREDIZATION OF BACTERIAL PROBE 5TO RNA OF TABLE 65 ORGANISMS FOUND IN URINE. HYBREDIZATION OF BACTERIAL PROBE 6 TO RNA OF % Probe ORGANISMS FOUND IN URINE Organism ATCC: Bound 55 % Probe Candida albicans 18804 1.8 Organism ATCC Bound Candida krusei 34135 1.7 Candida parapsilosis 22019 2.2 Candida albicans 18804 3.0 Candida tropicalis 750 1.8 Candida krusei 34135 2.0 Citrobacter freundi 8090 39 60 Candida parapsilosis 2201.9 2.2 Enterobacter aerogenes 13048 38 Citrobacter freundi 8090 54 Enterobacter cloacae 13047 43 Enterobacter aerogenes 13048 50 Escherichia coli 11775 31 Enterobacter cloacae 13047 58 Klebsielly oxytoca 13182 38 Escherichia coli 11775 63 Klebsiella pneumoniae 13883 66 Klebsiella oxytoca 13182 54 Morganella morgani 25830 50 65 Klebsiella pneumoniae 13883 55 Proteus mirabilis 2.9906 44 Morganella morgani 25830 60 Proteus vulgaris 13315 52 Proteus mirabilis 2.9906 64 5,547,842 61 62 TABLE 65-continued TABLE 67-continued HYBRIDIZATION OF BACTERIAL PROBE 6 TO RNA OF HYBRDIZATION OF BACTERIAL PROBET TO RNA ORGANISMS FOUND IN URINE OF ORGANISMS FOUND IN URINE % Probe % Probe Organism ATCCit Bound Organism ATCCit Bound Proteus vulgaris 13315 67 Torulopsis glabrata 2001 2.4 Providencia stuarti 2994 64 Ureaplasma urealyticum 27618 21 Pseudomonas aeruginosa 101.45 65 10 Pseudononas fluorescens 13525 31 Serratia marcescens 13880 67 Table 68 shows that probe 7 detects phylogenetically diverse Staphylococcus aureus 12600 53 Staphylococcus epidermidis 14990 34 bacteria and does not hybridize to human rRNA. Streptococcus agalactiae 13813 31 Streptococcus faecium 19434 18 TABLE 68 15 Torulopsis glabrata 2001 2.5 HYBRIDIZATION OF BACTERIAL PROBE 7TO RNAs OF ACROSS SECTION OF PHYLOGENETICALLY Table 66 shows that probe 6 detects some phylogenetically DIVERSE ORGANISMS diverse bacteria and does not hybridize to human rRNA. % Probe 20 Organism ATCC# Bound TABLE 66 Acinetobacter calcoaceticus 23055 86 HYBRDIZATION OF BACTERIAL PROBE 5TO RNAs Bacillus subtilis 6051 83 OF ACROSSSECTION OF PHYLOGENETICALLY Bacteroides fragilis 23745 69 DIVERSE ORGANISMS. Branhamella catarrhalis 25238 74 Campylobacter jejuni 33560 5.3 25 % Probe Chlamydia trachomatis VR878 4. Organism ATCC Bound Chromobacterium violaceum 290.94 69 clostridium perfringens 13124 68 Acinetobacter calcoaceticus 23055 73 Corynebacterium xerosis 373 23 Bacteroides fragilis 23745 7.0 Deinococcus radiodurans 35073 70 Branhamelia catarrhalis 25238 4.0 Dercia gummosa 15994 69 Deinococcus radiodurans 35073 5.5 30 Gardnerella vaginalis 14018 68 Derxia gunmosa 15994 3.0 Hafnia alvei 13337 77 Gardnerella vaginalis 14018 2.0 Moraxella osloensis 19976 68 Hafnia alvei 3337 3.5 Mycobacterium smegmatis 14468 64 Lactobacilius acidophilus 4356 17 Mycoplasma hominis 14027 4.0 Moraxella osloensis 19976 62 Neisseria gonorrhoeae 19424 53 Mycoplasma hominis 14027 44 35 Rahnella aquatis 33071 72 Rahnella aquatilis 33071 56 Rhodospirillum rubrum 11170 73 Yersinia enterocolitica 960 50 Vibrio parahaemolyticus 17802 67 Human 4.0 Yersinia enterocolitica 9610 66 Human 2.2

Table 67 shows that probe 7 hybridizes to the RNA of 40 bacteria commonly found in urine and does not detect yeast EXAMPLE 20 rRNA. Fungi encompass a morphologically and physiologically TABLE 67 diverse group of simple eucaryotic organisms. We estimate, HYBRIDIZATION OF BACTEREAL PROBE 7TO RNA 45 using published seque mces of three fungi, Neurospora OF ORGANISMS FOUND IN URINE crassav, Podospora, and Saccharomyces, that the rRNA of fungi are 58-60% homologous to E. coli and 84–90% % Probe homologous to one another. Some fungi grow as single cells Organism ATCCi Bound (yeasts), others as multinuclear filaments (molds) and still Candida albicans 18804 2.1 50 others can grow as either single cells or multicellular fila Candida krusei 34135 2.0 ments (dimorphic fungi). Although many fungi are harmless Candida tropicalis 750 2.2 inhabitants of their environments, others are harmful and Citrobacter freundi 8090 67 Enterobacter aerogenes 13048 69 cause disease. The presence of any fungi in some locations Enterobacter cloacae 13047 78 is undesirable or indicative of disease (e.g., culture media, Escherichia coli 11775 75 55 pharmaceutical products, body fluids such as blood, urine or Klebsiella oxytoca 13882 79 cerebrospinal fluid, and tissue biopsies). Low levels of fungi Klebsiella pneumonias 13883 77 Morganella morgani 25830 76 are considered acceptable in other products such as drinking Proteus mirabilis 2.9906 77 water and food products. This has created the need for a Proteus vulgaris 13315 79 means of detecting and quantitating fungi in a sample. Providencia stuartii 29914 64 60 Pseudomonas aeruginosa 101.45 76 The current methods for detecting and quantifying fungi Pseudomonas fluorescens 13525 78 involve microscopic examination of samples and culture on Serratia marcescens 13880 66 different media. Although most yeasts can be grown from Staphylococcus aureus 12600 71 Staphylococcus epidermidis 14990 75 clinical samples in a matter of days, some filamentous fungi Streptococcus agalactiae 13813 70 take up to four weeks culture time, after which special Streptococcus faecalis 19433 58 65 staining procedures, biochemical analysis and antigen tests Streptococcus faecium 19434 68 are performed. The oligonucleotide sequences below, when used in a hybridization assay, detect the five yeasts most 5,547.842 63 64 commonly isolated in the clinical setting, Candida albicans, probes were hybridized to a panel of bacteria most com Torulopsis glabrata, Candida tropicalis, Candida parapsi monly isolated from urine and shown not to react (Table 70). losis and Candida krusei. Five other fungi representing the Trichosporon, Blastomyces, Cryptococcus and Saccharomy Table71 shows that the probes do not hybridize to phylo ces genera are also detected. The present invention allows genetically diverse bacteria or to human RNA. one step detection of these organisms and, in relation to TABLE 69 culture, reduces the time to identification or elimination of these fungi as the cause of an infection. This represents a HYBREDIZATION OF YEAST PROBESTOYEAST RNA significant improvement over prior art methods. % Probe Bound The four probes which hybridize to the organisms of 10 interest were identified using 3 primers complementary to Organism ATCC 1 2 #3 i4 conserved regions on 18S or 28S rRNA. Sequence 1 was Blastomyces dermatitidis C.I. 25 14 15 5 obtained using an 18S primer with the sequence 5'-AGA Candida albicans 18804 40 63 56 2.0 ATTTCA CCT CTG-3'. Sequence 2 was obtained using a C. krusei 34135 73 62 2.2 70 28S primer with the sequence 5'-CCTTCT CCC GAA GTT 15 C. parapsilosis 2209 T1 63 65 2.0 C. tropicalis 750 62 71 71 2.0 ACG G-3'. Sequences 3 and 4 were obtained with a 28S Cryptococcus laurenti C.I. 43 1.4 1.5 1.5 primer with the sequence 5'-TTCCGACTT CCATGG CCA Cryptococcus neoformans C.I. 60 1.3 1.5 1.6 CCGTCC-3'. The following sequences were characterized Torulopsis glabrata 2001 61 44 62 2.0 and shown to hybridize to fungal rRNA. The sequences of Trichosporon beigelii C.I. 57 13 2.1 1.5 Saccharomyces cerevisiae, Saccharomyces carlsbergensis, 20 Saccharomyces cerevisiae C.I. 41 67 53 9 Escherichia coli and human rRNA were used for compari C.I. = Clinical isolate son with the sequences of interest.

1. CCC GAC CGT CCC TAT TAA TCA TTA CGA TGG 2. CGA CTT GGC ATG AAA ACT ATT CCT TCC TGT GG 3. GCT. CTT CAT TCA ATT GTC CAC GTT CAA TTA AGC AAC AAG G 4. GCT. CTG CAT TCA AAC GTC CGC GTT CAA TAA AGA AAC AGG G

Sequence 1, from 18S rRNA, is 30 bases in length and has aTm of 68° C. Sequence 2, from 23S rRNA, is 32 bases in length and has a Tm of 67° C. Sequence 3, from 23S rRNA, 35 is 40 bases in length and has a Tm of 66° C. Sequence 4, from 23S rRNA, is 40 bases in length and has a Tm of 68° C. Sequence 1 hybridizes in the region corresponding to position 845-880 of Saccharomyces cerevisiae 18s rRNA. Sequence 2 hybridizes in the region corresponding to posi 40 tion 1960-2000 of Saccharomyces cerevisiae 28s rRNA and sequences 3 and 4 hybridize in the region of 1225-1270 of the 28S rRNA. TABLE 70 To demonstrate the reactivity and specificity of these 45 probes for fungal RNA, they were used in hybridization HYBRIDIZATION OF FUNGAL PROBES 1-4TO RNA assays. 'P- or 'I-labeled oligonucleotide probes were OF ORGANISMS FOUND INURNE mixed with purified RNA or RNA released from cells by % Probe Bound standard lysis techniques in 0.2 ml of 0.48M sodium phos Organism ATCC 1 i2 i3 #4 phate pH 6.8, 1% sodium dodecyl sulfate, 1 mM EDTA, 1 50 mM EGTA and incubated at 60° C. for 2 hours. Following Citrobacter freundii 8090 5 1.7 15 2. incubation, 5 ml of 2% hydroxyapatite, 0.12M sodium Enterobacter aerogenes 13048 2.5 9 20 2.0 Enterobacter cloacae 13047 2.5 1.6 2.6 2.0 phosphate pH 6.8, 0.02% sodium dodecyl sulfate was added Escherichia coli 11775 3.0 2.0 16 15 and the samples incubated 10 minuteS. at 60° C. The Klebsiella oxytoca 13182 2.5 2.2 2.5 2.0 samples were centrifuged and the supernatants removed. 55 Klebsiella pneumonias 13883 2.5 2.2 2.1 2.0 Five ml of 0.12M sodium phosphate pH 6.8, 0.02% sodium Morgabella morgani 25830 2.0 2.8 1.7 1.9 Proteus mirabilis 2.9906 2.5 19 2.3 2.0 dodecyl sulfate was added, the samples were mixed, centri Proteus vulgaris 13315 2.0 2.2 2.0 15 fuged and the supernatants removed. The results are shown Providencia stuarti 29914 3.0 1.7 2.8 2.0 in Table 69. Probe 1 detects all ten fungi which were tested, Pseudomonas aeruginosa 1014.5 2.0 19 13 2.0 probe 2 detects all six of the yeasts which were tested, probe 60 Pseudomonas fluorescens 13525 2.5 2.7 2.1 2.0 Serratia marcescens 13880 2.5 1.7 1.8 2.0 3 detects five of the six yeasts, and probe 4 detects C. krusei Staphylococcus aureus 12600 2.0 1.7 .8 2.0 only. Thus probe 4 could be used to detect and identify C. Staphylococcus epidermidis 14990 3.0 1.5 3 2.0 krusei in samples, probe 1, 2 or combination of 3 and 4 could Streptococcus agalactiae 13813 2.5 1.9 13 2.5 be used to detect the yeasts, and probe 1 could be used to Streptococcus faecalis 19433 1.7 3.3 3.5 19 detect any of the ten organisms listed in Table 69. Streptococcus faecium 19434 20 2.9 2.1 15 One potential use for these probes is to identify yeasts in Ureaplasma urealyticum 27618 2. 3.1 24 1.8 urine samples or other normally sterile body fluids. The 5,547,842 65 66 tematics, Int'l J. System, Bacteriol, 37:463-464 (1987), the TABLE 71 phylogenetic definition of a species generally means 70% or HYBRIDIZATION OF FUNGAL PROBES 1-4 TO RNAs greater DNA:DNA homology. Despite the fact that these OF ACROSSSECTION OF PHYLOGENETICALLY organisms may be considered to be the same species under DIVERSE ORGANISMS established principles, we were able to make probes capable % Probe Bound of distinguising them. As expected, the rRNA homology between N. gonor Organism ATCCE it 2 3 i4 rhoeae and N. meningitidis is even greater because of known Acinetobacter calcoaceticus 23055 2.5 2.5 2.0 19 10 conserved regions. We noted a 1.0% difference between the Bacillus subtilis 6051 2.0 2.8 24 2.4 Bacteroides fragilis 23745 2.0 2.2 2.5 2.3 16S and a 1.1% difference between the 23S rRNA sequences Branhamella catarrhalis 25238 2.5 3.2 1.8 1.7 of N. gonorrhoeae and N. meningitidis using our sequencing Campylobacter jejuni 33560 2.5 2.1 2.0 19 data. Chlamydia trachomatis VR878 3.1 3.1 1.8 2.7 Making a probe for N. gonorrhoeae was complicated by Chronobacterium violaceum 29094 2.5 1.7 2.0 2.2 Clostridijim perfringens 13124 19 2.3 18 1.8 15 the fact that in some sites where N. meningitidis and N. Corynebacterium xerosis 373 1.6 4.8 1.8 1.1 gonorrhoeae differed, other Neisseria species were similar Deinmenectin radiodurans 35073 2.0 16 2.1 0.8 to N. gonorrhoeae. The few mismatches which exist Derxia gummosa 15994 3.0 15 1.7 1.8 Gardnerella vaginalis f 1401820 2.2 13 1.2 between these two species are in the most variable regions, Hafnia alvei 13337 10 2.5 1.7 16 i.e., regions which vary not only between species, but also Lactobacillus acidophilus 4356 2.0 2.7 2.0 1.9 20 from strain to strain. Despite the fact that some believed the Moraxella osloensis 19976 2.0 2.1 1.9 1.8 species could not be distinguished with nucleic acid probes Mycobacterium smegmatis 14468 1.6 1.8 1.8 1.7 Mycoplasma hominis 14027 1.5 18 1.6 1.5 at all, and others believed that rRNA was too conserved to Neisseria gonorrhoeae 10424. 2.0 2.7 1.6 16 be useful in probe diagnostics, we were able to make probes Rahnella aquatilis 33071 2.0 2.7 2.3 2.1 capable of differentiating N. gonorrhoeae and N. meningiti Rhodospirillum rubrum 1170 2.0 1.8 1.6 1.5 25 dis. Vibrio parahaemolyticus 17802 2.5 3.1 .7 1.6 Yersinia enterocolitica 9610 2.0 1.8 2.3 2.2 The present invention has significant advantages over Human 2.0 1.8 2.1 3.0 each of the prior art methods; the probes are more specific and much faster than culture methods. It also is believed that the probes are more sensitive, (i.e., able to detect a smaller CCCGACCGTCCCTATTAATCATTACGATGGTCCTAGAAAC CCCGACCGTCCCTATTAATCATTACGATGG

The first derivative works well at 65°C., the second at 60' 35 number of organisms in a clinical sample) than prior art C. methods. The primers used to identify these probe sequences had EXAMPLE 2 the following sequences: Gonorrhea is one of the most commonly reported bacte 1. GGCCGTTACCCCACCTACTASCTAAT rial infections in the United States, with over two million 40 2. GTATTACCGCGGCTGCTGGCAC cases reported annually. This sexually transmitted disease 3. GCTCGTTGCGGGACTTAACCCACCAT usually results in anterior urethritis in males and involves the Each of the rRNA sites chosen to target had at least two cervix in females. While severe complications and even mismatches to E. coli, N. menigitidis, N. cinerea, N. lac sterility can occur in untreated individuals, asymptomatic tamica, N. mucosa, and Kingella kingae. infections are common, resulting in carriers who unknow 45 Oligonucleotides complementary to sequences adjacent to ingly spread the disease. the probe regions were synthesized and used in the hydrid The causative agent, Neisseria gonorrhoeae, is a gram ization mix according to Hogan et al., U.S. Pat. No. 5,030, negative, oxidase positive diplococous with stringent growth 557; filed Nov. 24, 1987, entitled "Means and Method for requirements. The method used for diagnosis depends on the 50 Enhancing Nucleic Acid Hybridization (the "helper' patent site of infection and the patient symptoms. Gonococcal application). urethritis in males is diagnosed with good sensitivity and The following sequences were characterized and shown to specificity using gram stain. Culture, requiring 24–72 hours, be specific for Neisseria gonorrhoeae. The phylogenetically usually must be performed to confirm diagnosis of gonor nearest neighbors Neisseria meningitidis, N. lactamica, N. rhea from all females and asymptomatic males. Following cinerea, N. mucosa, and Kingella kingae were used for the detection of the organism from growth in culture, 55 comparison with the N. gonorrhoeae sequence. Neisseria gonorrhoeae must be identified by further tests 1. CCG CCGCTA CCC GGT AC such as carbohydrate degradation, coagglutination, fluores 2. TCA TCG GCC GCC GAT ATT GGC cent antibody screens or chromogenie enzyme substrate assays. 3. GAG CAT TCC GCA CAT GTC AAA ACC AGGTA 60 Sequence 1, complementary to 16S rRNA in the region Neisseria gonorrhoeae is particularly difficult to detect 125-150, is 17 bases in length and has a Tm of 56° C. and distinguish using a nucleic acid probe because it is very Sequence 2, complementary to 16S rRNA in the region closely related to N. meningitidis. Data published in Kings 455-485, is 21 bases in length and has a Tm of 63° C. bury, D. T., J. Bacteriol. 94:870-874 (1967) shows a Sequence 3, complementary to 16S rRNA in the region DNA:DNA homology for the two species of approximately 65 980-1015, is 29 bases in length and has a Tm of 57°C. 80–94%. Under guidelines established by the Ad Hoc Com The reactivity and specificity of the probes for Neisseria mittee on Reconciliation of Approaches to Bacterial Sys gonorrhoeae was demonstrated with a hybridization assay. 5,547,842 67 68 The three oligonuclotide probes were iodinated and mixed The present invention can be carried out on nonviral with unlabeled oligonucleotides of sequence 5'-CCC CTG organisms from purified samples or unpurified clinical CTTTCC CTCTCT AGA CGT ATG eGGTATTAG CTG samples such as sputum, feces, tissue, blood, spinal or ATC TTT CG-3',5'-GCCTTTTCTTCC CTG ACA AAA synovial fluids serum, urine or other bodily fluids, or other GTCCTTTACAAC CCG-3',5'-GGCACGTAGTTAGCC samples such as environmental or food samples. Prior to cell GGT GCT TAT TCT TCA GGT AC-3', and 5'-GGTTCT breakage and hybridization, the cells can be suspended or TCG CGT TGC ATC GAATTAATC CACATCATC CAC placed in solution. In the case of the unpurified samples CGC-3', and with purified RNA in 0.48M sodium phosphate, referred to above, the cells may remain intact and untreated ph6.8, 0.5% sodium dodecyl sulfate (SDS) and incubated at in their own biological environment prior to the assay. 60° C. for one hour. Following incubation, 4 ml of 2% The probes of the present invention may be used in an hydroxyapatite, 0.12M sodium phosphate pH 6.8, 0.02% 10 assay either alone or in combination with different probes. SDS was added and the mixture was incubated at 60° C. for Several individual probes also can be linked together during 5 minutes. The samples were centrifuged and the superna nucleic acid synthesis. This results in one probe molecule which contains multiple probe sequences, and therefore, tants were removed. Five ml of wash solution (0.12M multiple specificities. For example, a single nucleic acid sodium phosphate pH 6.8, 2% SDS) was added and the 15 molecule can be synthesized which contains both the Myco samples were mixed, centrifuged, and the supernatants bacterium avium and the Mycobacterium intracellulare removed. The amount of radioactivity bound to the sequences described in Examples 1 and 2. When hybridized hydroxyapatite was determined in a gamma counter. with either M.avium or M. intracellulare rRNA this probe Table 72 shows that the probes hybridize well to N. will hybridize completely. If the two probe sequences were gonorrhoeae RNA and do not hybridize to the other species 20 combined separately in an assay only one half of the mixed tested. individual probes will hybridize with either M. avium or M. intracellulare rRNA. Other embodiments also may be prac TABLE 72 ticed within the scope of the claims. For example, probes HYBRIDIZATION OF NEISSERIA GONORRHOEAE may be labelled using a variety of labels, as described PROBES 1-3 TONEISSERIA AND KINGELLARNAS 25 within, and may be incorporated into diagnostic kits. We claim: Organisms ATCC % Probe Bound 1. A hybridization assay probe able to detect the presence Kingella kingae 23332 0.09 of the Mycobacterium tuberculosis complex organisms Neisseria cinerea 14685 0.04 Mycobacterium africanum, Mycobacterium boris, and N. gonorrhoeae 19424 48.4 30 N. lactanica 23970 0.07 Mycobacterium tuberculosis, comprising an otigonucleotide N. meningitidis serogroup A 13077 0.04 10 to 100 nucleotides in length able to hybridize to a N. meningitidis serogroup B 13090 0.04 Mycobacterium tuberculosis complex nucleic acid target N. meningitidis serogroup C 13102 0.04 region present in each of Mycobacterium africamum, Myco N. mucosa 19696 0.07 N. subflava 14799 0.05 bacterium bovis, and Mycobacterium tuberculosis to form a 35 detectable target:probe duplex under selective hybridization assay conditions, said target region corresponding to, or The foliowing derivatives of Neisseria probes also have perfectly complementary to a nucleic acid corresponding to, been made and used: a region selected from the group consisting of: GAG GAT TCC GCA CAT GTC AAA ACC AGG bases 185-225 of E. coli 16S rRNA, 40 GAG GAT TCC GCA CAT GTC AAA ACC AGG TAA bases 540-575 of E. coli 23S rRNA, bases 1155-1190 of E. coli 23S rRNA, and CCC GCT ACC CGG TAC GTT C bases 2195-2235 of E. coli 23S rRNA, wherein said CCG CTA CCC GGT ACG TTC oligonucleotide comprises a sequence which is at least 45 75% complementary to a target sequence of 10 con tiguous nucleotides present in said target region in Although the above examples of performance were deter Mycobacterium africahum, Mycobacterium bovis, and mined using the standard assay format previously described, Mycobacterium tuberculosis, and said oligonucleotide the specific probes may be used under a wide variety of does not hybridize to nucleic acid from Mycobacterium experimental conditions. For example, additives may be 50 intracellulare or Mycobacterium avium to form a included to the reaction solutions to provide optimal reaction detectable non-target:probe duplex under said hybrid conditions for accelerated hybridization. Such additives may ization conditions. include buffers, chelators, organic compounds and nucleic 2. The probe of claim 1, wherein said oligonucleotide acid precipitating agents such as detergents, dihydroxyben comprises a sequence selected from the group consisting of: Zene, sodium dodecyl sulfate, sodium diisobutyl sulfosuc 55 5 TAAAGCGCTTTCCACCACAAGACATG cinate, sodium tetradecyl sulfate, sarkosyl and the alkali CATCCCGTG, metal salts and ammonium salts of SO-2A, PO-3, Cl- and 5' CCGCTAAAGCGCTTTCCACCACAAGA HC00-'. Such additives can be utilized by one skilled in the CATGCATCCCG art to provide optimal conditions for the hybridization 5 ACACCGCTAAAGCGCTTTCCACCACAA reaction to take place. These conditions for accelerated 60 hybridization of single stranded nucleic acid molecules into GACATGCATC, double stranded molecules are the subject of the above 5' CCATCACCACCCTCCTCCGGAGAGGAAAAGG, noted U.S. patent application Ser. No. 627,795 filed Jul. 5, 5 CTGTCCCTAAACCCGATTCAGGGTTC 1984, continuation filed Jun. 4, 1987 (serial no. not yet GAGGTTAGATGC, assigned) and Ser. No. 816,711 filed Jan. 7, 1986, which are 65 5' AGGCACTGTCCCTAAACCCGATTCAGGGTTC, both entitled ACCELERATED NUCLEICACID REASSO and sequences fully complementary and of the same length CIATION METHOD. thereto. 5,547,842 69 70 3. The probe of claim 1, wherein said target region assay conditions, said target region corresponding to, or corresponds to, or is perfectly complementary to a nucleic perfectly complementary to a nucleic acid corresponding to, acid corresponding to, bases 185-225 of E. coli 16S rRNA. a region selected from the group consisting of 4. The probe of claim 3, wherein said target region bases 185-225 of E. coli 16S rRNA, corresponds to bases 185-225 of E. coli 16S rRNA. 5. The probe of claim 3, wherein said target sequence of 5 bases 540-575 of E. coli 23S rRNA, 10 contiguous nucleotides is present in a nucleic acid bases 1155–1190 of E. coli 23S rRNA, and sequence selected from the group consisting of: bases 2195-2235 of E. coli 23S rRNA; wherein said oligonucleotide comprises a sequence which is at least 5' TAAAGCGCTTTCCACCACAAGACATG 75% complementary to a target sequence of 15 con CATCCCGTG, 10 tiguous nucleotides present in said target region in CCGCTAAAGCGCTTTCCACCACAAGA Mycobacterium africanum, Mycobacterium boris, and CATGCATCCCG, Mycobacterium tuberculosis, and said oligonucleotide ACACCGCTAAAGCGCTTTCCACCACAA does not hybridize to nucleic acid from Mycobacterium GACATGCATC and the sequences perfectly comple intracellulare or Mycobacterium avium to form a mcntary thereto. 15 detectable non-target:probe duplex under said hybrid 6. The probe of claim 1, wherein said target region ization conditions. corresponds to, or is perfectly complementary to a nucleic 19. The probe of claim 18, wherein said target region acid corresponding to, bases 540-575 of E. coli 23S rRNA. corresponds to, or is perfectly complementary to a nucleic 7. The probe of claim 6, wherein said target region acid corresponding to, bases 185-225 of E. coli 16S rRNA. 20 20. The probe of claim 19, wherein said target region corresponds to bases 540-575 of E. coli 23S rRNA. corresponds to bases 185-225 of E. coli 16S rRNA. 8. The probe of claim 6, wherein said 10 contiguous base 21. The probe of claim 19, wherein said target sequence region is present in a nucleic acid sequence selected from the of 15 contiguous nucleotides is present in a nucleic acid group consisting of: sequence selected from the group consisting of: 5' CCATCACCACCCTCCTCCGGAGAGGAAAAGG, 25 5' TAAAGCGCTTTCCACCACAAGACATG and the sequence perfectly complementary thereto. CATCCCGTG, 9. The probe of claim 1, wherein said target region 5' CCGCTAAAGCGCTTTCCACCACAAGA corresponds to, or is perfectly complementary to a nucleic CATGCATCCCG, acid corresponding to, bases 1155-1190 of E. coli 23S 5' rRNA. ACACCGCTAAAGCGCTTTCCACCACAA 30 GACATGCATC, and the sequences perfectly comple 10. The probe of claim 9, wherein said target region mentary thereto. corresponds to bases 1155-1190 of E. coli 23S rRNA. 22. The probe of claim 18, wherein said target region 11. The probe of claim 1, wherein said target region corresponds to, or is perfectly complementary to a nucleic corresponds to, or is perfectly complementary to a nucleic acid corresponding to, bases 540-575 of E. coli 23S rRNA. acid corresponding to, bases 2195-2235 of E. coli 23S 35 23. The probe of claim 22, wherein said target region rRNA. corresponds to bases 540-575 of E. coli 23S rRNA. 12. The probe of claim 11, wherein said target region 24. The probe of claim 22, wherein said 15 contiguous corresponds to bases 2195-235 of E. coli 23S rRNA. base region is present in a nucleic acid sequence selected 13. The probe of claim 11, wherein said target sequence from the group consisting of: of 10 contiguous nucleotides is present in a nucleic acid 5' CCATCACCACCCTCCTCCGGAGAGGAAA, and sequence selected from the group consisting of: 40 the sequence perfectly complementary thereto. 5' CTGTCCCTAAACCCGATTCAGGGTTC 25. The probe of claim 18, wherein said target region GAGGTTAGATGC, corresponds to, or is perfectly complementary to a nucleic acid corresponding to, bases 1155–1190 of E. coli 23S 5' AGGCACTGTCCCTAAACCCGATTCAGGGTTC, rRNA. and the sequences perfectly complementary thereto. 45 14. The probe of any of claims 3–3 wherein said oligo 26. The probe of claim 25, wherein said target region nucleotide comprises a sequence which is at least 90% corresponds to bases 1155–1190 of E. coli 23S rRNA. complementary to said target sequence of 10 contiguous 27. The probe of claim 18, wherein said target region nucleotides. corresponds to, or is perfectly complementary to a nucleic 15. The probe of claim 14, wherein said oligonucleotide 50 acid corresponding to, bases 2195-2235 of E. coli 23S. comprises a sequence which is 100% complementary to said rRNA. target sequence of 10 contiguous nucleotides. 28. The probe of claim 27, wherein said target region 16. The probe of claim 15, wherein said selective hybrid corresponds to bases 2195-2235 of E. coli 23S rRNA. ization assay conditions comprise 0.12M phosphate buffer 29. The probe of claim 27, wherein said 15 contiguous containing equimolar amounts of Na2HPO and NaH2PO, 55 base region is present in a nucleic acid sequence selected 1 mM EDTA and 0.02% sodium dodecyl sulfate at 65° C. from the group consisting of: 17. The probe of claim 14, wherein said oligonucleotide 5' CTGTCCCTAAACCCGATTCATTTCGTGT is 15-50 bases in length. TAGATGC, 18. A hybridization assay probe able to detect the presence 5. AGGCACTGTCCCTAAACCCGATTCAGGGTTC, of the Mycobacterium tuberculosis complex organisms 60 and the sequences perfectly complementary thereto. Mycobacterium africanurn, Mycobacterium bovis, and 30. The probe of any of claims 19–29, wherein said Mycobacterium tuberculosis, comprising an oligonucleotide oligonucleotide comprises a sequence which is at least 90% 15 to 100 nucleotides in length able to hybridize to a complementary to said target sequence of 15 contiguous Mycobacterium tuberculosis complex nucleic acid target nucleotides. region present in each of Mycobacterium africanum Myco 65 31. The probe of claim 30, wherein said oligonucleotide bacterium boris, and Mycobacterium tuberculosis to form a comprises a sequence which is 100% complementary to said detectable target:probe duplex under selective hybridization target sequence of 15 contiguous nucleotides. 5,547,842 71 72 32. The probe of claim 31, wherein said selective hybrid 40. The method of claim 39, wherein said target region ization assay conditions comprise 0.12M phosphate buffer corresponds to bases 540-575 of E. coil 23S rRNA. containing equimolar amounts of Na2HPO and NaH2PO4, 41. The method of claim 39, wherein said target sequence 1 mM/DTA and 0.02% sodium dodecyl sulfate at 65° C. of 10 contiguous nucleotides is present in a nucleic acid 33. The probe of claim 31, wherein said oligonucleotide sequence selected from the group consisting of: is 15-50 bases in length. 5' CCATCACCACCCTCCTCCGGAGAGGAAAAGG, 34. A method for determining whether a Mycobacterium and the sequence perfectly complementary thereto. tuberculosis complex organism may be present in a sample, 42. The method of claim 34, wherein said target region comprising the steps of: corresponds to, or is perfectly complementary to a nucleic a) providing to said sample an oligonucleotide able to 10 acid corresponding to, bases 1155-1190 of E. coli 23S hybridize to a Mycobacterium tuberculosis complex rRNA. nucleic acid target region present in each of Mycobac 43. The method of claim 42, wherein said target region terium africahum, Mycobacterium bovis, and Myco corresponds to bases 1155-1190 of E. coli 23S rRNA. bacterium tuberculosis to form a detectable target 44. The method of claim 34, wherein said target region :probe duplex under hybridization assay conditions, corresponds to, or is perfectly complementary to a nucleic said target region corresponding to, or perfectly 15 acid corresponding to, bases 2195-2235 of E. coli 23S complementary to a nucleic acid corresponding to, a rRNA. region selected from the group consisting of: 45. The method of claim 44, wherein said target region bases 185-225 of E. coli 16S rRNA, corresponds to bases 2195-2235 of E. coli 23S rRNA. bases 540-575 of E. coli 23S rRNA, 46. The method of claim 44, wherein said target sequence bases 1155-1190 of E. coli 23S rRNA, and 20 of 10 contiguous nucleotides is present in a nucleic acid bases 2195-2235 of E. coli 23S rRNA, wherein said oligonucleotide comprises a sequence which is at sequence selected from the group consisting of: least 75% complementary to a target sequence of 10 5' CTGTCCCTAAACCCGATTCAGGGTTC contiguous nucleotides present in said target region GAGGTTAGATGC, in Mycobacterium africahum, Mycobacterium bovis, 5' AGGCACTGTCCCTAAACCCGATTCAGGGTTC, and Mycobacterium tuberculosis, and said oligo and the sequences perfectly complementary thereto. nucleotide does not hybridize to nucleic acid from 47. The method of any of claims 35-46, wherein said Mycobacterium intracellulare or Mycobacterium oligonucleotide comprises a sequence which is at least 90% avium to form a detectable non-target:probe duplex complementary to said target sequence of 10 contiguous under said hybridization conditions, and 30 nucleotides. b) detecting hybridization of said probe to nucleic acid 48. The method of claim 47 wherein said oligonucleotide present in said sample under said hybridization condi comprises a sequence which is 100% complementary to said tions. target sequence of 10 contiguous nucleotides. 35. The method of claim 34, wherein said oligonucleotide 49. The method of claim 48, wherein said oligonucleotide comprises a sequence selected from the group consisting of: 35 is 15-50 bases in length. 50. A hybridization assay probe able to detect the presence 5' TAAAGCGCTTTCCACCACAAGACATG of the Mycobacterium tuberculosis complex organisms CATCCCGTG, Mycobacterium africanum, Mycobacterium bovis, and 5 CCGCTAAAGCGCTTTCCACCACAAGA Mycobacterium tuberculosis, comprising an oligonucleotide CATGCATCCCG 40 10 to 100 nucleotides in length able to hybridize to a 5' ACACCGCTAAAGCGCTTTCCACCACAA Mycobacterium tuberculosis complex nucleic acid target GACATGCATC, region present in each of Mycobacterium africanum, Myco 5' CCATCACCACCCTCCTCCGGAGACCAAAATGC, bacterium boris, and Mycobacterium tuberculosis to form a 5' CTGTCCCTAAACCCGATTCAGGGTTC detectable target:probe duplex under selective hybridization GAGGTTAGATGC, 45 assay conditions, said target region having the sequence 5' 5' AGGCACTGTCCCTAAACCCGATTCAGGGTTC, CCCTACCCACACCCACCACAAGGT or the complement and sequences fully complementary and of the same thereof, wherein said oligonucleotide comprises a sequence length thereto. which is at least 75% complementary to a target sequence of 36. The method of claim 34, wherein said target region 10 contiguous nucleotides present in said target region, and corresponds to, or is perfectly complementary to a nucleic 50 said oligonucleotide does not hybridize to nucleic acid from acid corresponding to, bases 185-225 of E. coli 16S rRNA. Mycobacterium intracellulare or Mycobacterium avium to 37. The method of claim 36, wherein said target region form a detectable non-target:probe duplex under said corresponds to bases 185-225 of E. coli 16S rRNA. hybridization conditions. 38. The method of claim 36, wherein said target sequence 51. The probe of claim 50, wherein said oligonucleotide 55 comprises a sequence which is at least 90% complementary of 10 contiguous nucleotides is present in a nucleic acid to said target sequence of 10 contiguous nucleotides. sequence selected from the group consisting of: 52. The probe of claim 51, wherein said oligonucleotide 5' TAAAGCGCTTTCCACCACAAGACATG comprises a sequence which is 100% complementary to said CATCCCGTG, target sequence of 10 contiguous nucleotides. 5 CCGCTAAAGCGCTTTCCACCACAAGA 60 53. A method for determining whether a Mycobacterium CATGCATCCCG, tuberculosis complex organism may be present in a sample, 5' ACACCGCTAAAGCGCTTTCCACCACAA comprising the steps of: GACATGCATC, and the sequences perfectly comple a) providing to said sample an oligonucleotide able to mentary thereto. hybridize to a Mycobacterium tuberculosis complex 39. The method of claim 34, wherein said target region 65 nucleic acid target region present in each of Mycobac corresponds to, or is perfectly complementary to a nucleic terium africamum, Mycobacterium bovis, and Myco acid corresponding to, bases 540-575 of E. coli 23S rRNA. bacterium tuberculosis to form a detectable target 5,547,842 73 74 :probe duplex under hybridization assay conditions, b) detecting hybridization of said probe to nucleic acid said target region having the sequence 5' CCCTAC present in said sample under said hybridization condi CCACACCCACCACAAGGT or the complement tions. thereof; wherein said oligonucleotide comprises a 54. The method of claim 53, wherein said oligonucleotide sequence which is at least 75% complementary to a comprises a sequence which is at least 90% complementary target sequence of 10 contiguous nucleotides present in to said target sequence of 10 contiguous nucleotides. said target region, and said oligonucleotide does not 55. The method of claim 54, wherein said oligonucleotide hybridize to nucleic acid from Mycobacrerium intrac comprises a sequence which is 100% complementary to said ellulare or Mycobacterium avium to form a detectable target sequence of 10 contiguous nucleotides. non-target:probe duplex under said hybridization con 10 ditions, and ; : : ck sk

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION 5,547,842 Page l of 7 PATENT NO. : DATED : August 20, 1996

INVENTOR(S) : James J. Hogan et al. it is certified that error appears in the above-indentified patent and that said Letters Patent is hereby corrected as shown below:

Column 1, line 26: Delete "non-vital" and insert-non-viral

Column 2, Line 26: Delete "non-vital" and insert-non-viral Column 3, Line 4: Delete "non-vital"and insert-non-viral

Column 11, Line 42: Delete "bobis" and insert-bovis

Column 11, Line 64: Delete "acidalcohol" and insert-acid alcohol-.

Column 11, Line 67: Delete "differenigate" and insert-differentiate

Column 14, Table 2, Under Heading ORGANISM, 25th organism from the top: Delete "M. trivials" and insert-M. triviale

Column 16, Table 6, Under Heading ORGANISM, 11th organism from the top: Delete "M. haemoplilum and insert - M. haemophilum

Column 17, Table 8, Under Heading ORGANISM, 17th organism from the top: Delete "Rhodospirillura" and insert -Rhodospirilium

Column 19, Table 11, Under Heading ORGANISM, 2nd organism from the top:

Delete "Fusobacterium bucleatum' and insert -Fusobacterium nucleatum

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION 5,547,842 Page 2 of 7 PATENT NO. : DATED : August 20, 1996 INVENTOR(S) : James J. Hogan et al. it is certified that error appears in the above-indentified patent and that said Letters Patent is hereby corrected as shown below:

Column 19, Line 49: Delete "characteristics and insert- characteristics.- Column 20, Line 48: Delete "Organisms" and insert -organisms

Column 25, Table 20, Under Heading % PROBE BOUND, 28tho rganism from the top,

M. Ulcerans: Delete "O. 17" and insert -0.7-

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION 5,547,842 Page 3 of 7 PATENT NO. : DATED : August 20, 1996 INVENTOR(S) : James J. Hogan et al. It is certified that error appears in the above-indentified patent and that said Letters Patent is hereby corrected as shown below: Column 31, Table 25, first Column, last row: Delete "29335". Column 32, Table 27, Under Heading ORGANISM, 3rd organism from the top: Delete "arcginini" and insert-arginini Column 36, Line 26: Delete "using" and insert -Using-. Column 39, Line7: Delete "66°C.." and insert -66°C.-- Column 39, Line 8: Delete "67°C.." and insert-67oC Column 39, Line 43: Delete "ufogenital" and insert -urogenital-. Column 41, Line 38: Delete "eteriris and insert --enteritis Column 42, Line 4: Delete "dCT" and insert-CCT Column 42, Line 34: Delete "supernatant" and insert --Supernatant--. Column 44, Line 63: Delete "pseudomas" and insert -Pseudomanas

Column 46, Line 39: Delete "cloacas" and insert --cloacae-.

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION 5,547,842 Page 4 of 7 PATENT NO. : DATED : August 20, 1996 INVENTOR(S) : James J. Hogan et al. it is certified that error appears in the above-indentified patent and that said Letters Patent is hereby corrected as shown below: Column 46, Line 56: Delete "238" and insert-23S Column 48, Line 18: Delete "dodeoyl" and insert --dodecyl-. Column 48, Line 26: Delete "Was" and insert --was-- Column 49, Line 40: Delete "selflimited" and insert-self-limited. Column 50, Line 18: Delete "standard." and insert-standard Column 50, Table 50, Under Heading ORGANISM, 13th organism from the top: Delete "saintpaut" and insert -Saintpaul--. Column 51, Table 50, Under Heading ORGANISM, 5th organism from the top: Delete "alamae" and insert-salamae Column 52, Line 59: Delete "TGAAAA" and insert-TGAAAA Column 52, Line 63: Delete "ocli" and insert --Coli-.

Column 54, Line 21: Delete "overPrior" and insert --Over prior--.

UNITED STATES PATENT ANDTRADEMARK OFFICE CERTIFICATE OF CORRECTION 5,547,842 Page 5 of 7 PATENT NO. : DATED : August 20, 1996

INVENTOR(S) :

James J. Hogan et al. it is certified that error appears in the above-indentified patent and that said Letters Patent is hereby corrected as shown below:

Column 54, Line 37: Delete "Hellobacterium" and insert-Heliobacterium--. Column 54, Line 42: Delete "S," and insert -S.--. Column 54, Line 42: Delete "guintana" and insert --quintana-.

Column 54, Line 56: Delete "ETCA" and insert --TCA-. Column 55, Line 4: Delete "folkowing" and insert-following-.

Column 60, Table 64, Under Heading ORGANISM, 14th organism from the top: Delete "oaloeksis" and insert --Osloensis--.

Column 62, Line 45: Delete "seque moes" and insert-sequences--. Column 62, Line 46: Delete "Crassav" and insert --Crassa--.

Column 63, Line 54: Delete "minuteS." and insert-minutes--. Column 64, Table 70, Under Heading ORGANISM, 7th organism from the top: Delete "morgabella" and insert-Morganella-. Column 65, Table 71, Under Heading ATCC#, Organism NEISSERIA GONORRHOEAE:

Delete "10424" and insert --19424--. Column 65, Line 34: Before "The" insert --Two derivatives of probe 1 also were made.--.

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION 5,547,842 Page 6 of 7 PATENT NO. : DATED : August 20, 1996 INVENTOR(S) : James J. Hogan et al. It is certified that error appears in the above-indentified patent and that said Letters Patent is hereby Corrected as shown below: Column 66, Line 39: Delete "GGCCGTTACCCCACCTACTASCTAAT" and insert-GGCCGTTACCCCACCTACTAGCTAAT-- Column 67, Line 3: Delete "eCG" and insert-CGG Column 67, Line 9: Delete "ph" and insert --pH-. Column 68, Line 29: Delete "africamum" and insert-africanum Column 68, Line 29: Delete "boris" and insert -bovis-. Column 68, Line 30: Delete "otigonucleotide" and insert-oligonucleotide Column 68, Line 33: Delete "africamum" and insert -africanum--. Column 68, Line 47: Delete "africahum" and insert --africanum Column 69, Line 38: Delete "2195-235" and insert --2195-2235 Column 69, Line 46: Delete "3-3" and insert -3-13 Column 69, Line 61: Delete "africanurn" and insert-africanum Column 69, Line 65: Delete "africanum" and insert -africanum,--. Column 69, Line 66: Delete "boris" and insert-bovis

UNITED STATES PATENT ANDTRADEMARK OFFICE CERTIFICATE OF CORRECTION 5,547,842 Page 7 of 7 PATENT NO. : DATED : August 20, 1996 INVENTOR(S) : James J. Hogan et al. it is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Column 70, Line 11: Delete "boris" and insert -bovis--. Column 70, Line 49: Delete "23S." and insert-23S Column 71, Line 4: Delete "mM/DTA" and insert-mM EDTA-. Column 71, Line 13: Delete "africahum" and insert -africanum Column 71, Line 25: Delete "africahum" and insert -africanum-. Column 72, Line 43: Delete "boris" and insert-bovis Column 72, Line 66. Delete "africamum" and insert --africanum--. Column 73, Line 8: Delete "Mycobacrerium" and insert --Mycobacterium Signed and Sealed this Thirtieth Day of June, 1998 Attest 4. (east

BRUCE LEHMAN Attesting Officer Commissioner of Patents and Trademarks